DOT1 histone methyltransferases as a target for identifying therapeutic agents for leukemia

ABSTRACT

The present invention provides polypeptides with histone H3 lysine 79 methyltransferase activity as well as nucleic acids encoding the same. Also provided are methods of using the polypeptides and nucleic acids of the invention in screening assays to identify compounds of interest. Further provided are diagnostic methods for leukemia and prognostic methods to predict the course of the disease in a subject.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.12/251,037, filed 14 Oct. 2008 (now U.S. Pat. No. 8,901,286), which is adivisional of U.S. application Ser. No. 10/866,908, filed 14 Jun. 2004(now U.S. Pat. No. 7,442,685), which claims the benefit of priority fromU.S. provisional patent application Ser. No. 60/478,497, filed 13 Jun.2003, the disclosures of which are incorporated herein by reference intheir entireties.

STATEMENT OF FEDERAL SUPPORT

This invention was made with federal government support under Grant Nos.GM063067 & GM068804 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing, size:36,880 bytes, filed Oct. 14, 2008, in parent U.S. application Ser. No.12/251,037, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel histone methyltransferases aswell as nucleic acids encoding the same; also disclosed are methods foridentifying compounds that modulate the activity of the histonemethyltransferase, methods of identifying compounds that inhibit bindingof a polypeptide to the histone methyltransferase, methods ofidentifying candidate compounds for the treatment of leukemia, anddiagnostic methods based on histone methylation.

BACKGROUND OF THE INVENTION

Higher-order chromatin structures are of profound importance in generegulation and epigenetic inheritance (Wu and Grunstein (2000) TrendsBiochem. Sci. 25:619-623). Post-translational modifications of corehistones critically influence the establishment and maintenance ofhigher-order chromatin structures. The unstructured tails of certaincore histones are extensively modified by acetylation, methylation,phosphorylation, ribosylation and ubiquitination. A “histone code”hypothesis, linking histone modifications to chromatin structures, hasbeen the focus of intensive recent studies (Strahl and Allis (2000) Mol.Cell. Biol. 22:1298-1306; Turner (2000) Bioessays 22:836-845). Histonemethylation has emerged as a major form of histone modification. (Strahland Allis (2000) Mol. Cell. Biol. 22:1298-1306; Zhang and Reinberg(2001) Genes Dev. 15:2343-2360). In particular, a large family of SETdomain-containing histone methyltransferases (HMTases) has beenidentified (Lachner and Jenuwein (2002) Curr. Opin. Cell Biol.14:286-298). SET domain proteins have been shown to methylate variousN-terminal lysine residues of histone H3 and H4. Histone lysinemethylation has been associated with diverse biological processesranging from transcriptional regulation to the faithful transmission ofchromosomes during the cell division (Grewal and Elgin (2002) Curr.Opin. Genet. Dev. 12:178-187).

Further, lysine methylation catalyzed by SET domain containing proteinshas been linked to cancer (Schneider, et al. (2002) Trends Biochem. Sci.27:396-402). For example, the H3-K4 methyltransferase MLL is frequentlytranslocated in leukemia (Ayton and Cleary (2001) Oncogene 20:5695-5707;Milne, et al. (2002) Mol. Cell 10:1107-1117; Nakamura, et al. (2002)Mol. Cell 10:1119-1128) and the H3-K27 methyltransferase EZH2 isoverexpressed in a number of tumors and its expression level correlateswith the invasiveness of these tumors (Bracken, et al. (2003) EMBO J.22:5323-5335; Kleer, et al. (2003) Proc. Natl. Acad. Sci. USA100:11606-11611; Varambally, et al. (2002) Nature 419:624-9).

Chromosomal translocation is one of the major causes of human cancer,particularly in acute leukemias. The most common chromosomerearrangement found in leukemia patients involves the mixed lineageleukemia gene MLL (also called ALL or HRX) located at 11q23 (Ayton andCleary (2001) Oncogene 20:5695-5707). MLL is the human homologue ofDrosophila Trithorax (Trx), a protein involved in maintaining the “onstate” of the homeotic box (Hox) gene expression during embryonicdevelopment. MLL contains a number of functional motifs including theN-terminal AT hook DNA binding motif and the C-terminal SET domainrequired for its H3-lysine 4 methyltransferase activity (Milne, et al.(2002) Mol. Cell 10:1107-1117; Nakamura, et al. (2002) Mol. Cell10:1119-1128). As a result of chromosome translocation, MLL N-terminibecome fused in-frame with one of more than 30 partner proteins (Aytonand Cleary (2001) Oncogene 20:5695-5707). Regardless of whether thefusion partner is normally localized to the nucleus or cytoplasm, thechimeras are always nuclear (Dimartino and Cleary (1999) Br. J.Haematol. 106:614-626). Given that the DNA binding domain of MLL isstill retained in the fusion proteins, the MLL target genes will bedifferentially regulated as a result of loss of the MLL SET domain andgain of fusion partner function in the chimeras. HOXA9 has emerged asone of the most relevant MLL target genes in human acute myeloidleukemia (AML) as it is always up-regulated in AML (Golub, et al. (1999)Science 286:531-537). Indeed, the leukemogenic potential of Hoxa9 wasdirectly demonstrated by the development of AML in mice receivingtransplantation of bone marrow cells overexpressing Hoxa9 (Kroon, et al.(1998) EMBO J. 17:3714-3725). Both Hoxa7 and Hoxa9 have been shown to berequired for MLL fusion proteins to transform myeloid progenitor cells(Ayton and Cleary (2003) Genes Dev. 17:2298-2307). However, themechanism by which different MLL fusion proteins up-regulate HOXA9 andhow higher levels of HOXA9 leads to leukemia remains to be elucidated.

Dot1 is an evolutionarily conserved protein that was originallyidentified in S. cerevisiae as a disruptor of telomeric silencing(Singer, et al. (1998) Genetics 150:613-632). It also functions at thepachytene checkpoint during the meiotic cell cycle (San-Segundo andRoeder (2000) Mol. Biol. Cell. 11:3601-3615). Sequence analysis of yeastDot1 revealed that it possesses certain characteristic SAM bindingmotifs, similar to the ones in protein arginine methyltransferases(Dlakic (2001) Trends Biochem. Sci. 26:405-407).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the first identification ofa post-translational modification within the globular domain of thehistone. In particular, the inventors have observed methylation oflysine 79 of histone H3 (“H3-K79”) and have identified a novel class ofhistone methyltransferase (HMTase) designated “DOT1” that methylatesH3-K79 in vivo. DOT1L (see, e.g., SEQ ID NO:2) is a member of the DOT1family. Similar to other HMTases, the DOT1 polypeptides contain aS-adenosylmethionine (SAM) binding site and use SAM as a methyl donor.However, unlike other reported HMTases, the DOT1 polypeptides do notcontain a SET domain.

Accordingly, as a first aspect, the present invention provides anisolated nucleic acid comprising a nucleotide sequence encoding a DOT1Lpolypeptide, the nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence comprising the nucleotide sequence of SEQID NO:1; (b) a nucleotide sequence that hybridizes to the completecomplement of a nucleotide sequence comprising the nucleotide sequenceof SEQ ID NO:1 under stringent conditions, wherein the nucleotidesequence encodes a polypeptide having histone H3 lysine 79 (H3-K79)methyltransferase activity; (c) a nucleotide sequence having at least95% nucleotide sequence similarity with the nucleotide sequence of SEQID NO:1, wherein the nucleotide sequence encodes a polypeptide havingH3-K79 methyltransferase activity; (d) a nucleotide sequence thatdiffers from the nucleotide sequence of (a) above due to the degeneracyof the genetic code; (f) a nucleotide sequence comprising a functionalfragment of SEQ ID NO:1, wherein the functional fragment comprises atleast the coding region of the histone methyltransferase catalyticdomain of SEQ ID NO:1 or a nucleotide sequence that hybridizes to thecomplete complement of the coding region of the histonemethyltransferase catalytic domain of SEQ ID NO:1 under stringentconditions, and wherein the functional fragment encodes a polypeptidehaving H3-K79 methyltransferase activity; (g) a nucleotide sequencecomprising a functional fragment of SEQ ID NO:1, wherein the functionalfragment comprises at least the coding region of the histonemethyltransferase catalytic domain of SEQ ID NO:1 or a nucleotidesequence having at least 95% nucleotide sequence similarity thereto, andwherein the functional fragment encodes a polypeptide having H3-K79methyltransferase activity; and (h) a nucleotide sequence comprising afunctional fragment of SEQ ID NO:1, wherein the functional fragmentcomprises at least the coding region of the histone methyltransferasecatalytic domain of SEQ ID NO:1 or a nucleotide sequence that differstherefrom due to the degeneracy of the genetic code, and wherein thefunctional fragment encodes a polypeptide having H3-K79methyltransferase activity.

Also provided are vectors comprising the nucleic acids of the invention,as well as cells comprising the inventive nucleic acids and vectors.

As a further embodiment, the invention provides an isolated DOT1Lpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of SEQ ID NO:2; (b) an aminoacid sequence having at least 95% amino acid sequence similarity withthe amino acid sequence of SEQ ID NO:2, wherein the DOT1L polypeptidehas histone H3 lysine 79 (H3-K79) methyltransferase activity; and (c) afunctional fragment of at least 400 amino acids of the amino acidsequence of (a) or (b) above, wherein the functional fragment comprisesa DOT1L histone methyltransferase catalytic domain and has H3-K79methyltransferase activity.

Also provided are fusion proteins comprising the inventive DOT1Lpolypeptide. Further provided are cells comprising the fusion proteinsof the invention.

As a further aspect, the invention comprises a method of identifying acompound that modulates DOT1L histone H3 lysine 79 (H3-K79)methyltransferase activity, the method comprising contacting a DOT1Lpolypeptide of the invention with a nucleosome substrate comprising H3in the presence of a test compound; detecting the level of H3-K79methylation of the nucleosome substrate under conditions sufficient toprovide H3-K79 methylation, wherein an elevation or reduction in H3-K79methylation in the presence of the test compound as compared with thelevel of histone H3-K79 methylation in the absence of the test compoundindicates that the test compound modulates DOT1L H3-K79methyltransferase activity.

As still a further aspect, the invention provides a method ofidentifying a candidate compound for the prevention and/or treatment ofleukemia, the method comprising: contacting a DOT1L polypeptide of theinvention with a nucleosome substrate comprising histone H3 in thepresence of a test compound; detecting the level of histone H3 lysine 79(H3-K79) methylation of the nucleosome substrate under conditionssufficient to provide H3-K79 methylation, wherein a reduction in H3-K79methylation in the presence of the test compound as compared with thelevel of H3-K79 methylation in the absence of the test compoundindicates that the test compound is a candidate compound for theprevention and/or treatment of leukemia.

As still a further aspect, the invention provides a method ofidentifying a compound that inhibits binding of DOT1L polypeptide to asecond polypeptide, comprising: contacting a DOT1L polypeptide accordingto the invention with a second polypeptide in the presence of a testcompound; detecting the level of binding between DOT1L polypeptide andthe second polypeptide under conditions sufficient for binding of DOT1Lpolypeptide to the second polypeptide, wherein a reduction in bindingbetween DOT1L polypeptide and the second polypeptide in the presence ofthe test compound as compared with the level of binding in the absenceof the test compound indicates that the test compound inhibits bindingof DOT1L polypeptide to the second polypeptide.

As yet another aspect, the invention provides a method of identifying acandidate compound for the prevention and/or treatment of leukemia,comprising: contacting a DOT1L polypeptide of the invention with AF10 oran MLL fusion protein in the presence of a test compound; detecting thelevel of binding between the DOT1L polypeptide and AF10 or MLL fusionprotein under conditions sufficient for binding of DOT1L polypeptide toAF10 or MLL fusion protein, wherein a reduction in binding between DOT1Lpolypeptide and AF10 or the MLL fusion protein in the presence of thetest compound as compared with the level of binding in the absence ofthe test compound indicates that the test compound is a candidatecompound for the prevention and/or treatment of leukemia.

As a further aspect, the invention provides a method of diagnosingwhether a subject has or is at risk for developing leukemia and/ordetermining the prognosis for the course of the disease, comprising:obtaining a biological sample comprising nucleosomes from a subject;detecting the level of histone H3 lysine 79 (H3-K79) methylationassociated with the HoxA9 gene; wherein an elevation in HoxA9-associatedH3-K79 methylation in the biological sample as compared with the levelof HoxA9-associated H3-K79 methylation in a non-leukemic biologicalsample is diagnostic that the subject has or is at risk of developingleukemia and/or is prognostic of the course of the disease in thesubject.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show that H3-K79 methylation is conserved from yeast tohuman. (A) Location of K79 of histone H3 relative to the two α-helices(α1 and α2) and loop 1 (L1) (SEQ ID NO:3) based on the known nucleosomestructure (Luger, et al. (1997) Nature 389:251-260). (B)Characterization of the H3-mK79 antibody. HeLa core histones,recombinant histone H3 that are not methylated, methylated by SET7 orSUV39 were analyzed by COOMASSIE® staining and western blot analysisusing the mK4-, mK9-, and mK79-specific antibodies. The mK79-specificantibody was raised against an H3 peptide containing di-methylated K79(Ile-Ala-Gin-Asp-Phe-^(m)Lys-Thr-Asp-Leu-Arg-Phe; SEQ ID NO:4). (C)H3-K79 methylation occurs in a wide range of organisms. Equivalentamounts of histones, as evidenced by COOMASSIE® staining, from theindicated organisms were analyzed by western blot analysis using theH3-mK79-specific antibody.

FIGS. 2A-2E show the identification of a human DOT1-like protein. (A)Alignment of the amino acid sequences of the DOT1 family proteins. Onlythe most conserved regions are shown. Sequences used in the alignmentinclude yeast DOT1 (NP_010728; SEQ ID NO:5), and its homologs from human(AF509504; SEQ ID NO:6), Drosophila (AAF54122; SEQ ID NO:7), and C.elegans (NP_510056; SEQ ID NO:8). Four additional putative C. elegansproteins in GENBANK (NP_509981, NP_490970, NP_509997, NP_508351) alsoshow homology to DOT1. Sequences predicted to be involved in SAM binding(Dlakic (2001) Trends Biochem. Sci. 26:405-407; incorporated byreference herein in its entirety for teachings of SAM binding domains inDOT1) are indicated. Numbers represent the amino acid number ofrespective proteins. Gaps are indicated by “−”. Amino acids that areidentical or have similar properties are boxed. (B) Amino acid sequenceof human DOT1L (SEQ ID NO:2). Predicted SAM binding motifs areunderlined. Mutation of the boxed amino acids abolished the HMTaseactivity of hDOT1L. (C-E) Nucleic acid sequence encoding human DOT1L(SEQ ID NO:1).

FIGS. 3A and 3B demonstrate that hDOT1L is a nucleosomal H3-K79-specificHMTase. (A) Recombinant hDOT1L is a nucleosomal H3-specific HMTase invitro. About 0.2 μg of different versions of recombinant hDOT1L proteinwere incubated with 10 μg of core histones or equivalent amounts ofnucleosomes using standard methods (Wang, et al. (2001) Mol. Cell8:1207-1217). The reaction mixtures were resolved in SDS-PAGE followedby COOMASSIE® staining and fluorogram analysis. Recombinant hDOT1Lproteins shown in the upper panel are 15-fold of what were used in theHMTase assay. (B) hDOT1L methylates H3-K79 in vivo. Empty vector, aswell as vectors that encode FLAG®-tagged wild-type or mutant (m) hDOT1Lwere transfected into 293T cells using the QIAGEN® EFFECTENE™Transfection Reagent. Two days after transfection, cells were collectedfor the preparation of total cell lysates and histones. Expression ofwild-type and mutant hDOT1L was verified by western blot analysis usinganti-FLAG® antibody. Equal loading of lysates was confirmed by probingfor tubulin. Equal loading of histones was verified by COOMASSIE®staining.

FIGS. 4A-4E show that the level of H3-K79 methylation is cell cycleregulated. (A) Cells released from double thymidine block were analyzedby flow cytometry. The numbers of cells (arbitrary units) were plottedagainst DNA content. (B) Cell extracts and histones derived from samplesanalyzed in (A) were analyzed by western blot analysis. SLBP and cyclinA were used as cell cycle markers, tubulin was used as loading control.Equal loading of histones were revealed by COOMASSIE® staining. Thelevel of H3-K79 methylation was analyzed by probing with themK79-specific antibodies. The cell cycle stage of each sample isindicated on top of the panel. ‘asy’ represents synchronized cellextracts. (C, D) Identical to (A, B) except that the cells were arrestedat M-phase before release. (E) mK79 level in relation to cell DNAcontent. Cells were labeled with FITC-conjugated goat anti-rabbitsecondary antibody in the absence (left panel) or presence of primaryantibody (middle and right panels) as indicated. DNA in each cell waslabeled with propidium iodide (PI). Each spot represents an individualcell. The numbers represent an arbitrary FITC unit. Unlabeled cells wereanalyzed by flow cytometry (left panel insert) to serve as standard forDNA content. The intensity of FITC signal (measurement of Mi-2 or mK79)is plotted against PI signal (measurement of DNA content that correlateswith cell cycle stages). Arrow heads and arrows represent early S-phaseand late S phase, respectively.

FIG. 5 shows that hDOT1L(1-416) is an active histone methyltransferase.Purified HeLa and chicken nucleosomes were used as substrates in theassay and proteins were analyzed by autoradiography.

FIGS. 6A-6D show HMTase activity and mono-nucleosome binding of hDOT1Lderivatives. (A) The positively charged region (amino acids 390-407) isimportant for the HMTase activity of hDOT1L. Equal amount of hDOT1Ldeletion mutants (upper panel), indicated on top of the panel, wereanalyzed for HMTase activity (middle panel) and the quantitation ispresented in the bottom panel. (B) The positively charged regionrequired for HMTase activity is also required for mono-nucleosomebinding. Mono-nucleosome binding of the hDOT1L deletions mutants wasanalyzed by gel mobility shift assays at three different proteinconcentrations (μM). (C) Amino acid sequence (amino acids 361-416; SEQID NO:9) of the C-terminal of hDOT1L(1-416) encompassing the chargedregion required for the HMTase activity. A stretch of similar aminoacids located at the N-terminal of yeast Dot1 is also shown (SEQ IDNO:10). Identical amino acids between the yeast and human proteins aresingle underlined, and similar amino acids are double underlined. (D)The HMTase activities of hDOT1L(1-416) and Δ(376-390) in the absence andpresence of increasing concentrations (μM) of a positively chargedcompetitor peptide were analyzed by autoradiograph (top panel) and thequantitation is shown at the bottom panel.

FIGS. 7A-7C show that hDOT1L is degraded extensively in cells. (A)Recombinant hDOT1L(1-351) digested by thrombin from GST-hDOT1L(1-352)was used to generate a rabbit polyclonal antibody. The antibody wasaffinity purified by incubating the serum with the antigen. Protein (100μg) derived from HeLa nuclear extracts (NE) or nuclear pellet (NP), orrecombinant GST-hDOT1L(1-351) were subjected to SDS-PAGE and blottedwith either preimmune serum or affinity-purified hDOT1L antibody. (B)Full-length hDOT1L exists only in the nucleus. N-terminal FLAG®-taggedor C-terminal HA-tagged hDOT1L was transfected into 293T cells. Proteins(100 μg) derived from nuclear or cytoplasm fractions of transfectedcells were subjected to western blot analysis using antibodies againstFLAG® or HA. (C) hDOT1L tagged with an N-terminal FLAG® and a C-terminalHA was transfected into U2OS cells. Localization of hDOT1L was viewed byimmunofluorescent staining using a monoclonal antibody against FLAG® anda polyclonal antibody against HA. Transfected cells with nuclearlocalization or cytoplasmic localization were counted and presented aspercentage of total transfected cells.

FIGS. 8A-8C show that hDOT1L is subjected to ubiquitination in vivo. (A)hDOT1L is subjected to ubiquitination but its degradation is not throughthe 26S proteasome pathway. FLAG®-tagged hDOT1L and Tat wereco-expressed with HA-ubiquitin in the presence or absence of proteasomeinhibitor MG132. Protein extracts derived from the transfected cellswere immunoprecipitated with anti-FLAG® antibody and blotted withantibody against FLAG®. (B) hDOT1L ubiquitination occurs in the regionbetween amino acid 469-756. Diagram of the hDOT1L constructs tested inthe ubiquitination assays described in A. Results of the assays aresummarized to the right of the panel. “++” represents full level ofpolyubiquitination; “+” represents lower level of polyubiquitination;“−” represents mono or no ubiquitination. (C) hDOT1L can beubiquitinated equally well in both nucleus and cytoplasm. Western blotof the ubiquitination assays of the two hDOT1L(1-767) constructsindicated. Lanes 5 and 6 indicate a similar level of ubiquitinationregardless of whether the protein is present in cytoplasm (lane 5) ornucleus (lane 6).

FIG. 9 illustrates that hDOT1L contains signals for nuclear andcytoplasm localization and presents a diagram of full-length anddeletion hDOT1L constructs. NLS1 and NLS2 represent two nuclearlocalization signals (NLS) identified by the PredictNLS program. Inaddition, a coiled-coil and a region conserved between Drosophila andhuman DOT1L (CR) is also indicated.

FIGS. 10A-10E show the identification of the three nuclear exportsignals (NESs) in hDOT1L. (A) Diagram of the different EGFP-hDPT1Lfusion constructs and summary of their nuclear exclusion patterns. “++”,“+”, and “−” represent strong, weak, and no nuclear exclusion,respectively. (B) Representative pictures of the constructs, shown in A,transfected into U2OS cells. (C) Top panel is a sequence alignment ofNES1 (SEQ ID NO:12) and NES2 (SEQ ID NO:13) of hDOT1L with NES ofprotein kinase A inhibitor (PKI; SEQ ID NO:11). Conserved amino acidsare underlined and amino acids mutated in NES1M are boxed. Bottom panelsshow mutation on the two C-terminal hydrophobic amino acids of NES1 (SEQID NO:14) disrupts NES1 function. (D) Demonstration of the NES1 functionin hDOT1L localization in the context of full-length protein. (E) Thefunction of each of the three NESs of hDOT1L can be blocked by LMBtreatment.

FIGS. 11A and 11B show the identification of AF10, a MLL fusion partnerin leukemia, as an hDOT1L associated protein. (A) Yeast two-hybridresults demonstrating that only yeast cells containing both hDOT1L andAF10 plasmids could grow and form a blue colony onSD-Trp-Leu/-His/-adenine/+X-α-Gal plates (“SD-W/-L/-H/-Ade/+X-α-Gal”).(B) AF10 and hDOT1L co-immunoprecipitate when co-expressed in 293Tcells. FLAG® antibody was used for immunoprecipitation, and HA antibodywas used for western blot analysis.

FIGS. 12A-12D show that the octapeptide motif and Leucine zipper (OM-LZ)region of AF10, required for leukemogenesis of MLL-AF10, is alsorequired for hDOT1L and AF10 interaction. (A) Diagram of AF10 functionalmotifs and sequence alignment of the OM and LZ regions. Arrow indicatesthe break point for MLL-AF10 fusion. Human AF10 (AY598745; SEQ IDNO:15), and its homologs from mouse (“mAF10”, O54826; SEQ ID NO:16),Drosophila, (“dAF10”, Alhambra, AAF72595; SEQ ID NO:17), and C. elegans(“cAF10”, Cezf, 2122400A; SEQ ID NO:18) were compared. Conserved aminoacids are underlined. Mutations on the amino acids marked by * disruptthe interaction between hDOT1L and AF10. (B) Mammalian two-hybridanalysis identified the OM-LZ region of AF10 which is sufficient formediating AF10 and hDOT1L interaction. (C) Co-immunoprecipitationexperiments demonstrating that the OM-LZ region of AF10 is necessary forthe AF10 and hDOT1L interaction. Western blot analysis shown on the leftpanel indicated that the wild-type and the deletion mutant proteins wereexpressed to a similar level. (D) Mammalian two-hybrid assaysdemonstrating that both the evolutionarily conserved octapeptide(Glu-Gin-Leu-Leu-Glu-Arg-Gln-Trp; SEQ ID NO:19) motif (OM) and theleucine zipper (LZ) of AF10 contribute to the interaction. The mutationsare marked by * in panel A.

FIGS. 13A and 138 show that a leucine rich region in hDOT1L mediates theAF10 and hDOT1L interaction. (A) Co-immunoprecipitation assay identifieda leucine rich region (amino acids 472-767) of hDOT1L to be importantfor AF10 and hDOT1L interaction. (B) Mammalian two-hybrid assaysconfirmed and further narrowed the interaction regions of hDOT1L toamino acids 500-630. In addition, mutagenesis studies demonstrated thatleucines 532 and 592 are both important for the interaction. Numbersindicate the amino acid number of hDOT1L.

FIGS. 14A-14C shows that bone marrow transformation by MLL-hDOT1Lrequires the HMTase activity and a coiled-coil region (amino acids416-670) of hDOT1L. (A) Schematic representation of the retroviraltransduction procedures. (B) Diagram of retroviral constructs expressingMLL, MLL-AF10, and MLL-hDOT1L. The numbers refer to the amino acidnumber of corresponding proteins. The HMTase defective mutant(containing GCG to RCR change) is marked by *. (C) Relative colonynumbers generated per 10⁴ transduced bone marrow cells in the first,second, and third round of plating. The various control and fusionproteins expressed are indicated. Data presented is the average of fourindependent experiments with error bars.

FIGS. 15A and 15B show that transduction of the HMTase-defective hDOT1Linto MLL-AF10 transformed bone marrow cells attenuates the proliferationability of the transformed cells. (A) Diagram depicting the assay. (B)Colony numbers generated from transduction of 10⁴ cells of the thirdround of MLL-AF10-transformed bone marrow cells with wild-type orHMTase-defective MLL-hDOT1L(1-670). Data presented is the average of twoindependent experiments with error bars.

FIGS. 16A-16C show MLL-hDOT1L(1-670) immortalizes murine myeloidprogenitors and uncharacterized cells. (A) Morphology of colonies formedin methylcellulose by MLL-AF10- and MLL-hDOT1L(1-670)-transduced cells.(B) Total and individual numbers of the two types of colonies formed ineach round transduced by MLL-hDOT1L(1-670). (C) FACS analysis of theearly myeloid markers, Mac1 and c-Kit, of the cells derived from secondround plating of MLL-AF10- or hDOT1L(1-670)-transduced cells. Percent ofcells expressing the indicated surface antigens is indicated.

FIGS. 17A and 17B show a comparison of Hox gene expression patternsbetween primary bone marrow cells and MLL-AF10- orMLL-hDOT1L(1-670)-transduced cells. (A) RT-PCR showing expressionresponse to MLL-AF10 or MLL-hDOT1L(1-670) transduction for each of theHoxA genes and other Hox genes. The expression patterns of the two Meis1isoforms (a, b correspond to the upper and lower bands of the doublet,respectively) were also analyzed due to the reported collaborative roleof Hoxa and Meis genes in myeloid leukemogenesis (Nakamura, et al.(1996) Nat. Genet. 12:149-153). GAPDH serves as a control for equalinput in RT-PCR for different samples. Presence of MLL-AF10 orMLL-hDOT1L transgenes in the transformed cells was verified by PCR. “B”refers to primary bone marrow; “I” and “II” refer to the two differenttypes of colonies arising from MLL-hDOT1L(1-670) transduction; “A”refers to colonies arising from MLL-AF10 transduction; “N” is a negativecontrol for RT-PCR. (B) Summary of the genes overexpressed in transducedcells relative to none-transduced bone marrow cells analyzed in A.

FIGS. 18A and 18B depict the relationship between hDOT1L, AF10, andMLL-AF10 in normal (A) and leukemia (B) cells.

DETAILED DESCRIPTION OF THE INVENTION

Chromatin structure is important in gene regulation and epigeneticinherence. It is known that post-translational modifications of histonesare involved in the establishment and maintenance of higher-orderchromatin structure; further, it has been reported that the tails ofcertain core histones are modified by acetylation, methylation,phosphorylation, ribosylation and ubiquitination. The present inventionis based, in part, on the first identification of a post-translationalmodification within the globular domain of the histone. In particular,the inventors have observed methylation of lysine of histone H3(“H3-K79”) and have identified a novel class of histonemethyltransferase (HMTase) designated “DOT1” that methylates H3-K79 invivo. DOT1L is a member of the DOT1 HTMase family (see, e.g., SEQ IDNO:2). Similar to other HMTases, the DOT1 polypeptide contain aS-adenosylmethionine (SAM) binding site and use SAM as a methyl donor.However, unlike other reported HMTases, the DOT1 polypeptides do notcontain a SET domain.

The yeast homolog of DOT1 was originally identified as a Disruptor OfTelomeric silencing (the protein and nucleic acid sequences of yeastDOT1 can be found at Accession No. NP_010728; incorporated herein byreference in its entirety). The inventors have cloned and isolated thehuman DOT1 homolog, designated as hDOT1L (human DOT1-like protein), anddetermined that hDOT1L is an HMTase. The sequences of the human nucleicacid (SEQ ID NO:1) and protein (SEQ ID NO:2) have been deposited underGenBank accession number AF509504. Only the approximately 360 N-terminalamino acids of hDOT1L share significant sequence similarity with theyeast DOT1. The inventors have further identified DOT1 homologs from C.elegans (C. elegans, GenBank Accession number NP_510056 and CAA90610),Drosophila (GenBank Accession No. CG10272 and AAF54122), mouse (GenBankAccession No. XP_125730), Anopheles gambiae (GenBank Accession No.EAA03558), and Neurospora crassa (GenBank Accession No. EAA33634) fromsequences in public databases (the disclosures of which are incorporatedby reference herein in their entireties). The SAM binding domain amongthese homologs is conserved (approximately 30-100% amino acid sequenceidentity and 50-100% amino acid similarity [i.e., identical or conservedamino acids]; see FIG. 2A).

The 2.5 Å resolution structure of a fragment of the hDOT1L proteincontaining the catalytic domain (amino acids 1-416) has been solved. Theatomic coordinates for amino acids 1-416 of hDOT1L have been determinedand deposited in the RCSB database under ID code 1NW3 (see also, Min, etal. (2003) Cell 112:711-723), the disclosures of which are incorporatedherein by reference in their entireties.

The present invention will now be described with reference to theaccompanying drawings, in which preferred embodiments of the inventionare shown. This invention can be embodied in different forms and shouldnot be construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

As used in the description of the invention and the appended claims, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Except as otherwise indicated, standard methods can be used for theproduction of recombinant and synthetic polypeptides, fusion proteins,antibodies or antigen-binding fragments thereof, manipulation of nucleicacid sequences, production of transformed cells, and the like accordingto the present invention. Such techniques are known to those skilled inthe art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORYMANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989): F. M. AUSUBEL et al.CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates,Inc. and John Wiley & Sons, Inc., New York).

I. DOT1L Polypeptides and Nucleic Acids.

As one aspect, the present invention provides DOT1L polypeptides. Asused herein, the term “polypeptide” includes both proteins and peptides.The term “DOT1L polypeptide” is intended to encompass the DOT1Lpolypeptides specifically described herein (e.g., SEQ ID NO:2) as wellas functional equivalents thereof that have substantially similar aminoacid sequences (as described below) to the DOT1L polypeptidesspecifically described herein (e.g., SEQ ID NO:2) and have one or moreof the functional properties (also discussed below) of the DOT1Lpolypeptides specifically described herein. The term “DOT1L polypeptide”also encompasses functional fragments of the full-length DOT1Lpolypeptides specifically disclosed herein (e.g., SEQ ID NO:2) andfunctional equivalents thereof that have substantially similar aminoacid sequences to a fragment of a full-length DOT1L polypeptidespecifically disclosed herein (e.g., a fragment of SEQ ID NO:2) and haveone or more of the functional properties of the DOT1L polypeptidesspecifically disclosed herein.

By “functional” it is meant that the DOT1L polypeptide has the same orsubstantially similar H3-K79 HMTase activity, SAM binding activity,histone and/or nucleosome binding activity, AF10 binding activity,AF10-MLL or other MLL fusion protein binding activity, leukemogenicactivity and/or any other biological activity of interest as comparedwith a full-length DO1L polypeptide (e.g., SEQ ID NO:2). To illustrate,in representative embodiments, a functionally equivalent DOT1Lpolypeptide or functional DOT1L fragment has at least about 50%, 75%,85%, 90%, 95% or 98% or more of the relevant biological activity(ies) asthe DOT1L polypeptide of SEQ ID NO:2. Methods of assessing DOT1L bindingto histones, nucleosomes, nucleic acids or polypeptides can be carriedout using standard techniques that will be apparent to those skilled inthe art (see the Examples for exemplary methods). Such methods includeyeast and mammalian two-hybrid assays and co-immunoprecipitationtechniques.

Other biological activities associated with DOT1L such as H3-K79 HMTaseand leukemogenic activity can also be evaluated using standard methodsknown in the art, for example, as described in the Examples below.

In particular embodiments of the invention, the DOT1L polypeptide hasH3-K79 specific HMTase activity. By H3-K79 “specific” HMTase activity itis meant that all, or essentially all, of the HMTase activity isdirected to H3-K79 (e.g., using a histone or nucleosome substrate).

In particular embodiments of the invention, the functionally equivalentDOT1L polypeptide or functional DOT1L fragment has the same orsubstantially similar H3-K79 HMTase activity as the DOT1L polypeptide ofSEQ ID NO:2 or the catalytically active fragment of amino acids 1-416 ofSEQ ID NO:2. Methods of evaluating HMTase activity are known in the art,and are described in the Examples below. In representative embodiments,the functionally equivalent DOT1L polypeptide or functional DOT1Lfragment has at least about 50%, 75%, 85%, 90%, 95% or 98% or more ofthe H3-K79 HMTase activity as the DOT1L polypeptide of SEQ ID NO:2 orthe catalytically active fragment of amino acids 1-416 of SEQ ID NO:2.

An “isolated” polypeptide as used herein means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide. In particularembodiments, the “isolated” polypeptide is at least about 1%, 5%, 10%,25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure(w/w). In other embodiments, an “isolated” polypeptide indicates that atleast about a 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold,10,000-fold, or more enrichment of the protein (w/w) is achieved ascompared with the starting material.

The DOT1L polypeptides of the invention can be derived from any speciesof interest (e.g., mammalian [including but not limited to human,non-human primate, mouse, rat, lagomorph, bovine, ovine, caprine,porcine, equine, feline, canine, etc.], insect, yeast, avian, plants,etc.) as well as allelic variations, isoforms, splice variants and thelike. The DOT1L sequences can further be wholly or partially synthetic.

In particular embodiments, the DOT1L polypeptide comprises, consistsessentially of, or consists of an isolated DOT1L polypeptide of SEQ IDNO:2 or a functional fragment or functional equivalent thereof.

Functional equivalents of the DOT1L polypeptides of the inventionencompass those that have substantial amino acid sequence similarity,for example, at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% ormore amino acid sequence similarity with the amino acid sequencesspecifically disclosed herein (e.g., SEQ ID NO:2) or a functionalfragment thereof. Alternatively, nucleic acids encoding the DOT1Lpolypeptides of the invention have at least about 60%, 70%, 75%, 80%,85%, 90%, 95%, 97% or more nucleotide sequence similarity with thenucleotide sequences encoding the DOT1L polypeptides specificallydisclosed herein (e.g., SEQ ID NO:1) or a fragment thereof and encode afunctional DOT1L polypeptide.

In alternate embodiments, nucleic acids encoding the DOT1L polypeptidesof the invention have substantial nucleotide sequence similarity withthe DOT1L nucleic acids specifically disclosed herein (e.g., SEQ IDNO:1) or fragments thereof and hybridize to the nucleic acid sequencesdisclosed herein (e.g., SEQ ID NO:1) or fragments thereof under standardconditions as known by those skilled in the art and encode a functionalDOT1L polypeptide (including functional fragments).

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%Formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% Formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% Formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to the nucleicacids encoding the DOT1L polypeptides (including functional fragmentsthereof) disclosed herein. See, e.g., Sambrook et al., MolecularCloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring HarborLaboratory).

Further, it will be appreciated by those skilled in the art that therecan be variability in the nucleic acids that encode the DOT1Lpolypeptides of the present invention due to the degeneracy of thegenetic code. The degeneracy of the genetic code, which allows differentnucleic acid sequences to code for the same protein, is well known inthe art.

Likewise, those skilled in the art will appreciate that the presentinvention also encompasses fusion proteins (and nucleic acid sequencesencoding the same) comprising the DOT1L polypeptides (includingfunctional fragments) of the invention. For example, it may be useful toexpress the DOT1L polypeptides as a fusion protein that can berecognized by a commercially available antibody (e.g., FLAG motifs) oras a fusion protein that can otherwise be more easily purified (e.g., byaddition of a poly-His tail). Additionally, fusion proteins that enhancethe stability of the protein may be produced, e.g., fusion proteinscomprising maltose binding protein (MBP) or glutathione-S-transferase.As another alternative, the fusion protein can comprise a reportermolecule. In other particular embodiments, the DOT1L fusion protein is aDOT1L-MLL fusion (see, e.g., Example 8). DOT1L fusion proteins can alsobe generated for use in yeast two-hybrid systems (e.g., GAL4-DOT1Lfusions), as known in the art.

It will further be understood that the DOT1L polypeptides specificallydisclosed herein will typically tolerate substitutions in the amino acidsequence and substantially retain biological activity. To routinelyidentify polypeptides of the invention other than those specificallydisclosed herein, amino acid substitutions may be based on anycharacteristic known in the art, including the relative similarity ordifferences of the amino acid side-chain substituents, for example,their hydrophobicity, hydrophilicity, charge, size, and the like. Inparticular embodiments, conservative substitutions (i.e., substitutionwith an amino acid residue having similar properties) are made in theamino acid sequence encoding the DOT1L polypeptide.

In making amino acid substitutions, the hydropathic index of amino acidscan be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (see, Kyte and Doolittle, (1982) J. Mol. Biol.157:105; incorporated herein by reference in its entirety). It isaccepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, Id.),and these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(incorporated herein by reference in its entirety) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or polypeptide has sequence identity orsimilarity to a known sequence. One example of a useful algorithm is theBLAST algorithm, described in Altschul et al., J. Mol. Biol. 215,403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90,5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2program which was obtained from Altschul et al., Methods in Enzymology,266, 460-480 (1996). WU-BLAST-2 uses several search parameters, whichare preferably set to the default values. The parameters are dynamicvalues and are established by the program itself depending upon thecomposition of the particular sequence and composition of the particulardatabase against which the sequence of interest is being searched;however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., (1997) Nucleic Acids Res. 25, 3389-3402.

The CLUSTAL program can also be used to determine sequence similarity.This algorithm is described by Higgins et al. (1988) Gene 73:237;Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) NucleicAcids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; andPearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

The DOT1L polypeptides of the present invention also encompassfunctional DOT1L polypeptide fragments (e.g., having HMTase activity,having SAM binding activity, histone or nucleosome binding activity,AF10 binding activity, AF10-MLL or any other MLL fusion protein bindingactivity, leukemogenic activity and/or any other biological activity ofinterest), and functional equivalents thereof. The length of the DOT1Lfragment is not critical. Illustrative functional DOT1L proteinfragments comprise at least about 80, 100, 200, 300, 400, 500, 600, 700,800, 1000, 1200, 1400 or more amino acids (optionally, contiguous aminoacids) of a DOT1L polypeptide. The invention also provides nucleic acidsencoding the functional DOT1L fragments. Exemplary nucleic acidsencoding functional DOT1L fragments comprise at least about 250, 300,400, 500, 600, 700, 800, 1000, 1500, 2000, 2500, 3000, 4000 or morenucleotide bases (optionally, contiguous bases) of a nucleic acidencoding a full-length DOT1L polypeptide.

In particular embodiments, the invention provides functional DOT1Lfragments comprising the catalytic domain comprising the SAM bindingdomain (as well as adjacent sequences) and nucleic acids encoding thesame. In representative embodiments, the functional DOT1L fragmentcomprises a catalytically active fragment comprising amino acids 157 to270 of SEQ ID NO:2 or a functional equivalent thereof. In otherembodiments, the DOT1L fragment comprises a catalytically activefragment comprising amino acids 121-306 or amino acids 1-416 of SEQ IDNO:2 or a functional equivalent of either of the foregoing.

The functional DOT1L fragment comprising the catalytic domain canoptionally further comprise the DOT1L positively charged region (e.g.,amino acids 390 to 407 of SEQ ID NO:2 or a functional equivalentthereof).

In other embodiments, the functional fragment comprises the DOT1Lleucine rich interaction domain with AF10 (e.g., amino acids 500-630 ofSEQ ID NO:2 or a functional equivalent thereof). As a furtherembodiment, the functional fragment comprises the leucine zipper regionat amino acids 564-590 of SEQ ID NO:2 and/or the coiled coil region atamino acids 561-660 of SEQ ID NO:2 or functional equivalents of eitherof the foregoing.

In still other embodiments, the invention provides a functional DOT1Lfragment comprising a nuclear export signal. In illustrativeembodiments, the nuclear export signal comprises amino acids 482-496 ofSEQ ID NO:2 or a functional equivalent thereof, amino acids 600-635 ofSEQ ID NO:2 or a functional equivalent thereof and/or amino acids636-650 of SEQ ID NO:2 or a functional equivalent thereof. In otherembodiments, the nuclear export signal comprises amino acids 472-767 ofSEQ ID NO:2 or a functional fragment thereof.

Those skilled in the art will appreciate that functional DOT1L fragmentscan comprise two or more of the functional regions discussed above.

In yet a further embodiment, the functional fragment comprises theN-terminal portion of a DOT1 polypeptide (e.g., SEQ ID NO:2), forexample, approximately the N-terminal 10, 20, 30, 40, 50, 60, 70, 80, 90or 100 amino acids. In other embodiments, the functional fragment istruncated at the N-terminus, e.g., less than about 100, 85, 75, 60, 50,35, 20, 15, 10 or 5 amino acids are truncated from the N-terminus.

The invention also provides isolated nucleic acids encoding the DOT1Lpolypeptides of the invention. The nucleic acid can be DNA, RNA orchimeras thereof, single stranded or double-stranded, and can be fullyor partially synthetic or naturally occurring. The nucleic acids cancomprise modified nucleotides or nucleotide analogs as are well-known inthe art. Further, the nucleic acid can be from any species of origin,including but not limited to mammalian species such as human, non-humanprimate, mouse, rat, rabbit, cattle, goat, sheep, horse, pig, dog, cat,etc. and avian species.

In particular embodiments, the nucleic acid is an isolated nucleic acid.As used herein, an “isolated” nucleic acid means a nucleic acidseparated or substantially free from at least some of the othercomponents of the naturally occurring organism, such as for example, thecell structural components or other polypeptides or nucleic acidscommonly found associated with the nucleic acid.

In representative embodiments, the invention provides an isolated DOT1Lpolypeptide comprising an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of SEQ ID NO:2; (b) an aminoacid sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%,97% or more amino acid sequence similarity with the amino acid sequenceof SEQ ID NO:2, wherein the DOT1L polypeptide has H3-K79methyltransferase activity; and (c) a functional fragment of at leastabout 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1200, 1400 or moreamino acids of the amino acid sequence of (a) or (b) above, wherein thefunctional fragment comprises a DOT1L histone methyltransferasecatalytic domain and has H3-K79 methyltransferase activity.

In other illustrative embodiments, the invention provides an isolatednucleic acid comprising a nucleotide sequence encoding a DOT1Lpolypeptide, the nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence comprising the nucleotide sequence of SEQID NO:1; (b) a nucleotide sequence that hybridizes to the completecomplement of a nucleotide sequence comprising the nucleotide sequenceof SEQ ID NO:1 under stringent conditions (as defined above), whereinthe nucleotide sequence encodes a polypeptide having H3-K79methyltransferase activity; (c) a nucleotide sequence having at leastabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more nucleotide sequencesimilarity with the nucleotide sequence of SEQ ID NO:1, wherein thenucleotide sequence encodes a polypeptide having H3-K79methyltransferase activity; (d) a nucleotide sequence that differs fromthe nucleotide sequence of (a) above due to the degeneracy of thegenetic code; (f) a nucleotide sequence comprising a functional fragmentof SEQ ID NO:1, wherein the functional fragment comprises at least thecoding region of the histone methyltransferase catalytic domain of SEQID NO:1 or a nucleotide sequence that hybridizes to the completecomplement coding region of the histone methyltransferase catalyticdomain of SEQ ID NO:1 under stringent conditions, and wherein thefunctional fragment encodes a polypeptide having H3-K79methyltransferase activity; (g) a nucleotide sequence comprising afunctional fragment of SEQ ID NO:1, wherein the functional fragmentcomprises at least the coding region of the histone methyltransferasecatalytic domain of SEQ ID NO:1 or a nucleotide sequence having at leastabout 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more nucleotide sequencesimilarity thereto, and wherein the functional fragment encodes apolypeptide having H3-K79 methyltransferase activity; and (h) anucleotide sequence comprising a functional fragment of SEQ ID NO:1,wherein the functional fragment comprises at least the coding region ofthe histone methyltransferase catalytic domain of SEQ ID NO:1 or anucleotide sequence that differs therefrom due to the degeneracy of thegenetic code; and wherein the functional fragment encodes a polypeptidehaving H3-K79 methyltransferase activity.

As yet a further embodiment, the invention provides antisenseoligonucleotides and siRNA that hybridize to the DOT1L nucleic acids ofthe invention (e.g., under stringent hybridization conditions as definedabove) and inhibit production of DOT1L polypeptide. RNAi is a mechanismof post-transcriptional gene silencing in which double-stranded RNA(dsRNA) corresponding to a coding sequence of interest is introducedinto a cell or an organism, resulting in degradation of thecorresponding mRNA. The RNAi effect persists for multiple cell divisionsbefore gene expression is regained. RNAi is therefore a powerful methodfor making targeted knockouts or “knockdowns” at the RNA level. RNAi hasproven successful in human cells, including human embryonic kidney andHeLa cells (see, e.g., Elbashir et al., Nature (2001) 411:494-8). In oneembodiment, silencing can be induced in mammalian cells by enforcingendogenous expression of RNA hairpins (see Paddison et al., (2002), PNASUSA 99:1443-1448). In another embodiment, transfection of small (e.g.,21-23 nt) dsRNA specifically inhibits nucleic acid expression (reviewedin Caplen, (2002) Trends in Biotechnology 20:49-51).

The mechanism by which RNAi achieves gene silencing has been reviewed inSharp et al, (2001) Genes Dev 15: 485-490; and Hammond et al., (2001)Nature Rev Gen 2:110-119).

RNAi technology utilizes standard molecular biology methods. Toillustrate, dsRNA corresponding to all or a part of a target codingsequence to be inactivated can be produced by standard methods, e.g., bysimultaneous transcription of both strands of a template DNA(corresponding to the target sequence) with T7 RNA polymerase. Kits forproduction of dsRNA for use in RNAi are available commercially, e.g.,from New England Biolabs, Inc. Methods of transfection of dsRNA orplasmids engineered to make dsRNA are routine in the art.

Silencing effects similar to those produced by RNAi have been reportedin mammalian cells with transfection of a mRNA-cDNA hybrid construct(Lin et al., (2001) Biochem Biophys Res Commun 281:639-44), providingyet another strategy for silencing a coding sequence of interest.

In particular embodiments, the invention provides antisenseoligonucleotides and siRNA that have at least about 60%, 70%, 80%, 85%,90%, 95%, 98%, 99% or 100% sequence identity with the DOT1L nucleicacids described above. Methods of making and using antisenseoligonucleotides and siRNA are known in the art. There is no particularupper or lower limit to the antisense oligonucleotide or siRNA.Illustrative antisense oligonucleotides or siRNA will be about 8, 10,12, 15, 18, 20, 25, 30, 40, 50, 60, 75, 90, 100, 125 or 150 or morebases in length.

The invention also provides vectors, including expression vectors andgene delivery vectors, comprising the nucleic acids of the invention.Suitable vectors include bacterial expression vectors, fungal expressionvectors, mammalian vectors, yeast expression vectors and plantexpression vectors. Exemplary vectors include bacterial artificialchromosomes, cosmids, yeast artificial chromosomes, phage, plasmids,lipid vectors and viral vectors (e.g., adenovirus, adeno-associatedvirus, retrovirus, baculovirus, and the like).

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, yeast cells, insect cells(e.g., in the baculovirus expression system) or mammalian cells. Somesuitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecI (Baldari et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of nucleic acidsto produce proteins in cultured insect cells (e.g., Sf 9 cells) includethe pAc series (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) andthe pVL series (Lucklow, V. A., and Summers, M. d. (1989) Virology170:31-39).

Examples of mammalian expression vectors include pCDM8 (Seed, (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).

The vector generally comprises an expression control element (e.g., apromoter) operably associated with the nucleic acids of the invention.It will be appreciated that a variety of expression control elements canbe used depending on the level and tissue-specific expression desired.Further, the promoter can be constitutive or inducible (e.g., themetalothionein promoter or a hormone inducible promoter). The expressioncontrol element can be native or foreign to the host cell and can be anatural or a synthetic sequence. The promoter is generally chosen sothat it will function in the target cell(s) of interest. The nucleicacids can further be associated with other appropriate expressioncontrol sequences, e.g., transcription/translation control signals andpolyadenylation signals. Viral regulatory elements are often employed inmammalian cells. For example, commonly used promoters in mammalianexpression vectors are derived from polyoma, adenovirus 2,cytomegalovirus and Simian Virus 40.

Moreover, specific initiation signals are generally required forefficient translation of inserted protein coding sequences. Thesetranslational control sequences, which can include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic. Further provided are host cells (e.g., yeast,bacterial, mammalian, insect, plant or fungal cells) comprising theisolated nucleic acids and vectors of the invention. The cell can betransiently or stably transformed with the nucleic acid or vector of theinvention. In particular embodiments, a host cell is transiently orstably (e.g., by stable integration into the genome of the cell or by astably maintained episome) transformed with a nucleic acid of theinvention.

Further, the cell can be cultured (i.e., isolated) or can be a cell insitu in a living organism.

Still another aspect of the invention is a transgenic non-human animal(e.g., non-human mammal) comprising a recombinant nucleic acid encodinga DOT1L polypeptide or a functional fragment thereof. In alternativeembodiments, the transgenic non-human animal comprises a recombinantnucleic acid encoding an antisense oligonucleotide or siRNA thatinhibits production of DOT1L polypeptide. Generally, the transgenicnon-human animal will be stably transformed with the nucleic acid, e.g.,by stable integration into the genome of the animal. The transgenicnon-human animal can be of any species of interest including but notlimited to mouse, rat, guinea pig, rabbit, pig, horse, goat, sheep, cow,cat, dog, monkey, hamster, chicken and the like. Methods of makingtransgenic non-human animals are known in the art.

As yet a further embodiment, the invention provides antibodies andantibody fragments that specifically bind to the DOT1L polypeptides ofthe invention.

The term “antibody” or “antibodies” as used herein refers to all typesof immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodycan be monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep or human,or can be a chimeric antibody. See, e.g., Walker et al., Molec. Immunol.26, 403-11 (1989). The antibodies can be recombinant monoclonalantibodies produced according to the methods disclosed in U.S. Pat. No.4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also bechemically constructed according to the method disclosed in U.S. Pat.No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science 254,1275-1281).

Monoclonal antibodies used to carry out the present invention can beproduced in a hybridoma cell line according to the technique of Kohlerand Milstein, (1975) Nature 265, 495-97. For example, a solutioncontaining the appropriate antigen can be injected into a mouse and,after a sufficient time, the mouse sacrificed and spleen cells obtained.The spleen cells are then immortalized by fusing them with myeloma cellsor with lymphoma cells, typically in the presence of polyethyleneglycol, to produce hybridoma cells. The hybridoma cells are then grownin a suitable medium and the supernatant screened for monoclonalantibodies having the desired specificity. Monoclonal Fab fragments canbe produced in E. coli by recombinant techniques known to those skilledin the art. See, e.g., W. Huse, (1989) Science 246, 1275-81.

Antibodies specific to the target polypeptide can also be obtained byphage display techniques known in the art.

Methods of Use.

The DOT1L polypeptides and nucleic acids of the invention can be used ina number of research, diagnostic and/or therapeutic applications.

To illustrate, the inventive DOT1L polypeptides and nucleic acids can beused to screen for compounds that bind to and/or modulate (e.g.,increase or decrease) one or more biological activities of DOT1L,including but not limited to H3-K79 HMTase activity, SAM bindingactivity, histone and/or nucleosome binding activity, AF10 bindingactivity, AF10-MLL or other MLL fusion protein binding activity,leukemogenic activity and/or any other biological activity of interest.As described above, the DOT1L polypeptide can be a functional fragmentof a full-length DO1L polypeptide or functional equivalent thereof andcan comprise any DOT1 domain of interest, including but not limited tothe catalytic domain, the SAM binding domain and/or the positivelycharged domain, the AF10 interaction domain and/or a nuclear exportsignal.

In particular embodiments, the compound enhances, elevates, increases(or similar terms) one or more biological activities of DOT1L (e.g., byat least about 25%, 50%, 75%, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold,20-fold or more). In other embodiments, the compound is an inhibitorthat reduces, inhibits, decreases (or similar terms) of one or morebiological activities of DOT1L (e.g., by at least about 25%, 40%, 50%,60%, 75%, 80%, 85%, 90%, 95%, 98% or more). In particular embodiments,an inhibitory compound results in no detectable level of the DOT1Lbiological activity(ies) of interest.

In one particular embodiment, the invention provides a method ofidentifying a compound that modulates (e.g., inhibits or enhances) DOT1Lpolypeptide binding to a histone or nucleosome substrate comprising H3,the method comprising: contacting a DOT1L polypeptide with a histonenucleosome substrate comprising H3 in the presence of a test compound;detecting the binding of the DOT1L polypeptide to the substrate underconditions sufficient for binding of the DOT1L polypeptide to thesubstrate, wherein an elevation or reduction in DOT1L polypeptidebinding to the substrate in the presence of the test compound ascompared with the level of binding in the absence of the test compoundindicates that the test compound modulates binding of DOT1L polypeptideto a histone or nucleosome substrate comprising H3.

In another embodiment, the invention provides a method of identifying acompound that modulates a biological activity (as described above) of aDOT1L polypeptide, the method comprising: contacting a DOT1L polypeptidewith a histone or nucleosome substrate comprising H3 in the presence ofa test compound; detecting the level of the DOT1L activity of interestunder conditions sufficient for the DOT1L polypeptide to exhibit therelevant biological activity, wherein an elevation or reduction in theDOT1L activity in the presence of the test compound as compared with thelevel in the absence of the test compound indicates that the testcompound modulates DOT1L biological activity.

In a further embodiment, the invention provides a method of identifyinga compound that modulates DOT1L H3-K79 HMTase activity, the methodcomprising: contacting a DOT1L polypeptide with a histone or nucleosomesubstrate comprising H3 in the presence of a test compound; detectingthe level of H3-K79 methylation of the histone or nucleosome substrateunder conditions sufficient to provide H3-K79 methylation, wherein anelevation or reduction in H3-K79 methylation in the presence of the testcompound as compared with the level of histone H3-K79 methylation in theabsence of the test compound indicates that the test compound modulatesDOT1L H3-K79 HMTase activity.

The inventors have discovered that DOT1L has a number of bindingpartners including AF10 and MLL fusion proteins (e.g., AF10-MLL), whichare implicated in leukemogenesis (described in more detail below). Thus,as another aspect, the invention provides a method of identifying acompound that inhibits binding of DOT1L polypeptide to a secondpolypeptide, the method comprising: contacting a DOT1L polypeptide ofthe invention with a second polypeptide in the presence of a testcompound; detecting the level of binding between DOT1L polypeptide andthe second polypeptide under conditions sufficient for binding of DOT1Lpolypeptide to the second polypeptide, wherein a reduction in bindingbetween DOT1L polypeptide and the second polypeptide in the presence ofthe test compound as compared with the level of binding in the absenceof the test compound indicates that the test compound inhibits bindingof DOT1L polypeptide to the second polypeptide.

In particular embodiments, the second polypeptide is AF10 (includingAF10 fragments comprising the OM-LZ domain, e.g., amino acids 719-800 ofAF10 [human AF10 sequence, Accession No. AY598745; mouse AF10 sequence,Accession No. 054826]; the disclosures of which are incorporated hereinby reference in their entireties). In other representative embodiments,the second polypeptide is an MLL fusion protein (e.g., an MLL-AF10fusion protein or an MLL-ENL, MLL-ELL or MLL-CBP fusion protein).

In other particular embodiments, the DOT1L polypeptide (includingfunctional fragments) comprises amino acids 500 to 630 of SEQ ID NO:2 oran amino acid sequence having at least 95% amino acid sequencesimilarity thereto.

The present invention further provides methods of identifying compoundsfor the treatment and/or prevention of leukemia by identifying compoundsthat bind to and/or modulate the biological activity (as describedabove) of a DOT1 polypeptide.

In one embodiment, the invention provides a method of identifying acandidate compound for the prevention and/or treatment of leukemia, themethod comprising: contacting a DOT1L polypeptide of the invention witha histone or nucleosome substrate comprising histone H3 in the presenceof a test compound; detecting the level of H3-K79 methylation of thesubstrate under conditions sufficient to provide H3-K79 methylation,wherein an inhibition of H3-K79 methylation in the presence of the testcompound as compared with the level of H3-K79 methylation in the absenceof the test compound indicates that the test compound is a candidatecompound for the prevention and/or treatment of leukemia.

The invention further provides a method of identifying a candidatecompound for the prevention and/or treatment of leukemia, comprising:contacting a DOT1L polypeptide of the invention with AF10 or an MLLfusion protein; detecting the level of binding between the DOT1Lpolypeptide and AF10 or MLL fusion protein under conditions sufficientfor binding therebetween, wherein a reduction in binding between DOT1Lpolypeptide and AF10 or the MLL fusion protein in the presence of thetest compound as compared with the level of binding in the absence ofthe test compound indicates that the test compound is a candidatecompound for the prevention and/or treatment of leukemia.

In particular embodiments, the MLL fusion protein is an MLL-AF10 fusionprotein or an MLL-ENL, MLL-ELL or MLL-CBP fusion protein.

In other representative embodiments, the DOT1L polypeptide (includingfunctional fragments) comprises the DOT1L AF10 interaction domain, e.g.,amino acids 500-630 of SEQ ID NO:2 or a functional equivalent thereof.Alternatively, DOT1L polypeptides comprising the leucine zipper regionat amino acids 564-590 of SEQ ID NO:2 and/or the coiled coil region atamino acids 561 to 660 of SEQ ID NO:2 or functional equivalents ofeither of the foregoing can be used as targets to identify compoundsthat disrupt interactions between DOT1L and AF10 or an MLL fusionprotein.

By the terms “treating leukemia” or “treatment of leukemia”, it isintended that the severity of the disease is reduced. By the term“prevention of leukemia” or “preventing leukemia” it is intended thatthe methods at least partially eliminate or reduce the incidence oronset of leukemia. Alternatively stated, by treating or preventingleukemia (or grammatical variations thereof), it is meant that themethods and compounds of the invention slow, control, decrease thelikelihood or probability, or delay the onset of leukemia in thesubject.

Methods of assessing DOT1L binding to histones, nucleosomes, nucleicacids or polypeptides can be carried out using standard techniques thatwill be apparent to those skilled in the art (see the Examples forexemplary methods). Such methods include yeast and mammalian two-hybridassays and co-immunoprecipitation techniques.

Other biological activities associated with DOT1L such as H3-K79 HMTaseand leukemogenic activity can also be evaluated using standard methodsknown in the art, for example, as described in the Examples below.

The screening methods of the invention can be carried out in acell-based or cell-free system. As a further alternative, the assay canbe performed in a whole animal (including transgenic non-human animals).Further, with respect to cell-based systems, the DOT1L polypeptide (orany other polypeptide used in the assay) can be added directly to thecell or can be produced from a nucleic acid in the cell. The nucleicacid can be endogenous to the cell or can be foreign (e.g., agenetically modified cell).

Any compound of interest can be screened according to the presentinvention. Suitable test compounds include small organic compounds(i.e., non-oligomers), oligomers or combinations thereof, and inorganicmolecules. Suitable organic molecules can include but are not limited topolypeptides (including enzymes, antibodies and Fab′ fragments),carbohydrates, lipids, coenzymes, and nucleic acid molecules (includingDNA, RNA and chimerics and analogs thereof) and nucleotides andnucleotide analogs. In particular embodiments, the compound is anantisense nucleic acid, an siRNA or a ribozyme that inhibits productionof DOT1L polypeptide.

Small organic compounds (or “non-oligomers”) include a wide variety oforganic molecules, such as heterocyclics, aromatics, alicyclics,aliphatics and combinations thereof, comprising steroids, antibiotics,enzyme inhibitors, ligands, hormones, drugs, alkaloids, opioids,terpenes, porphyrins, toxins, catalysts, as well as combinationsthereof.

Oligomers include oligopeptides, oligonucleotides, oligosaccharides,polylipids, polyesters, polyamides, polyurethanes, polyureas,polyethers, and poly (phosphorus derivatives), e.g. phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones,sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C, H, N, O or S, and combinations thereof. Sucholigomers may be obtained from combinatorial libraries in accordancewith known techniques.

Further, the methods of the invention can be practiced to screen acompound library, e.g., a combinatorial chemical compound library (e.g.,benzodiazepine libraries as described in U.S. Pat. No. 5,288,514;phosphonate ester libraries as described in U.S. Pat. No. 5,420,328,pyrrolidine libraries as described in U.S. Pat. Nos. 5,525,735 and5,525,734, and diketopiperazine and diketomorpholine libraries asdescribed in U.S. Pat. No. 5,817,751), a polypeptide library, a cDNAlibrary, a library of antisense nucleic acids, and the like, or anarrayed collection of compounds such as polypeptide and nucleic acidarrays.

The invention also encompasses compounds identified by the screeningmethods described above.

As still a further aspect, the invention encompasses diagnostic methodsfor leukemia and/or prognostic methods for predicting the future courseof leukemia in a subject by assessing H3-K79 histone methylation,wherein an elevation in H3-K79 methylation as compared with a normal(e.g., non-leukemic) subject is diagnostic of leukemia and/or prognosticof the course of the disease. In particular embodiments, the pattern ofhistone H3 methylation is assessed, e.g., the level of H3-K79methylation and the level of H3-K4 methylation are determined (e.g., ata particular location of the HoxA9 gene), wherein an elevation in H3-K79methylation and/or a reduction in H3-K4 methylation as compared with anormal (e.g., non-leukemic) subject is diagnostic of leukemia and/orprognostic of the course of the disease. This embodiment of theinvention can be practiced with any mammalian subject including but notlimited to human, non-human primate, cattle, sheep, goat, cat, dog, pig,horse, rat, mouse, rabbit or guinea pig subjects. In particularembodiments, the subject has or is believed to be at risk of developingleukemia. In other embodiments, the subject is an animal model ofleukemia.

A “diagnostic method”, as used herein, refers to a screening procedurethat is carried out to identify those subjects that are affected with aparticular disorder.

A “prognostic method” refers to a method used to help predict, at leastin part, the course of a disease (e.g., more aggressive or lessaggressive). Alternatively stated, a prognostic method may be used toassess the severity of the disease. For example, the screening proceduredisclosed herein may be carried out to both identify an affectedindividual, to evaluate the severity of the disease, and/or to predictthe future course of the disease. Such methods may be useful inevaluating the necessity for therapeutic treatment, what type oftreatment to implement, and the like. In addition, a prognostic methodmay be carried out on a subject previously diagnosed with a particulardisorder when it is desired to gain greater insight into how the diseasewill progress for that particular subject (e.g., the likelihood that aparticular patient will respond favorably to a particular drugtreatment, or when it is desired to classify or separate patients intodistinct and different sub-populations for the purpose of conducting aclinical trial thereon).

It will be appreciated by those skilled in the art that the diagnosticand prognostic methods of the invention may not be conclusive and mayneed to be supplemented with other diagnostic and/or prognostic methodsto make a final diagnosis or prognosis.

One exemplary method of diagnosing whether a subject has or is at riskfor developing leukemia and/or determining the prognosis for the courseof the disease comprises: obtaining a biological sample comprisingnucleosomes from a subject; detecting the level of H3-K79 methylation inthe biological sample; wherein an elevation in H3-K79 methylation in thebiological sample as compared with the level of H3-K79 methylation in anon-leukemic biological sample is diagnostic that the subject has or isat risk of developing leukemia and/or prognostic of the course of thedisease in the subject. The method can optionally further includedetermination of the level of H3-K4 methylation, wherein a decrease inH3-K4 methylation in the biological sample as compared with anon-leukemic sample is diagnostic that the subject has or is at risk ofdeveloping leukemia and/or prognostic of the course of the disease inthe subject.

According to another representative embodiment, the invention provides amethod of diagnosing whether a subject has or is at risk for developingleukemia and/or determining the prognosis for the course of the disease,the method comprising: obtaining a biological sample comprisingnucleosomes from a subject; detecting the level of H3-K79 methylationassociated with one or more HoxA genes (e.g., the HoxA9 gene) in thebiological sample; wherein an elevation in HoxA gene-associated H3-K79methylation (e.g., associated with the HoxA9 gene) in the biologicalsample as compared with the level in a non-leukemic biological sample isdiagnostic that the subject has or is at risk of developing leukemiaand/or prognostic of the course of the disease in the subject. Themethod can optionally further include determination of the level ofH3-K4 methylation associated with one or more HoxA genes (e.g., theHoxA9 gene), wherein a decrease in HoxA gene associated H3-K4methylation in the biological sample as compared with a non-leukemicsample is diagnostic that the subject has or is at risk of developingleukemia and/or prognostic of the course of the disease in the subject.

In exemplary embodiments, an “elevation” or “increase” (or similarterms) in methylation (e.g., H3-K79 methylation) is at least about a25%, 50%, 75%, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold or moreincrease in methylation as compared with the level of methylation in anon-leukemic biological sample.

In other representative embodiments, a “decrease” or “reduction” (orsimilar terms) in methylation (e.g., H3-K4 methylation) is at leastabout a 25%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or moredecrease in methylation as compared with the level of methylation in anon-leukemic biological sample. In particular embodiments, nomethylation is detectable.

Any suitable biological sample can be used including cell or tissuesamples, umbilical cord samples, blood, plasma or serum samples, urineor fecal samples, mucus or sputum samples, and the like. In particularembodiments, the biological sample is a B cell or bone marrow sample. Inother representative embodiments, the biological sample is a histone ornucleosome preparation comprising histone H3 (e.g., obtained from Bcells or bone marrow cells). In representative embodiments, cells ortissue are removed from a subject, cultured and nucleosomes preparedfrom the cultured cells or tissue.

By “non-leukemic biological sample” it is meant a suitable controlsample that is indicative of a normal subject (i.e., not having or atrisk for developing leukemia). For example, the sample can be isolatedfrom a normal subject or, in some instances (e.g., a nucleosomepreparation), can be isolated from cultured cells.

As further uses of the present invention, the DOT1L nucleic acids andpolypeptides can be used in methods of methylating histones (H3) in acell free system, in cultured cells or in vivo. In particularembodiments, the substrate is a histone substrate comprising H3. Inother embodiments, the substrate is a nucleosome substrate comprisingH3. Other uses of the DOT1L nucleic acids and DOT1L polypeptides as wellas antisense and siRNA molecules that inhibit DOT1L polypeptideproduction include modulation of telomeric silencing, regulation of geneexpression and/or cell cycle regulation.

Having described the present invention, the same will be explained ingreater detail in the following examples, which are included herein forillustration purposes only, and which are not intended to be limiting tothe invention.

Example 1 Experimental Procedures HMTase-Mediated Methylation ofH3-Lysine 79

Antibodies, hDOT1L Constructs and Protein Preparation.

The methyl-K79-specific antibody was raised by injection of rabbits witha synthetic peptide coding for amino acids 74-84 of histone H3 with K79di-methylated (Ile-Ala-Gln-Asp-Phe-mLys-Thr-Asp-Leu-Arg-Phe; SEQ IDNO:4). The methyl-K4, -K9 antibodies were purchased from UpstateBiotechnology (Lake Placid, N.Y.). The full-length hDOT1L was derivedfrom two over-lapping EST clones BF507396 and BF982417. The FLAG®-taggedconstructs were cloned into the EcoRI/XhoI sites of a pcDNA-derivedvector by PCR. The GST-fusion of the hDOT1L N-terminal fragments werealso cloned into the EcoRI/XhoI sites of pGEX-KG vector by PCR. Themutants were generated through PCR-based mutagenesis. All PCR-generatedconstructs were verified by sequence analysis. The GST-hDOT1L fusionproteins were purified as according to standard methods (Wang, et al.(2001) Mol. Cell 8:1207-1217). The recombinant proteins were thencleaved using thrombin following manufacturer's instructions. Proteinswere quantified by COOMASSIE® staining.

Cell Synchronization, Labeling and Flow Cytometry.

To obtain cells synchronized at the G1/S boundary, HeLa cells weretreated with 2 mM thymidine (SIGMA, St. Louis, Mo.) for 18 hours,followed by a 9 hour release in thymidine-free medium, and then treatedagain with 2 mM thymidine for 17 hours to arrest cells at the beginningof S phase. The synchronized cells were released in fresh medium andharvested every two hours. HeLa cells synchronized at the mitotic stagewere prepared by blocking with 2 mM thymidine for 18 hours, releasingfor 3 hours, and then incubating with 100 ng/mL nocodazole for 12 hours.The cells were then washed three times with phosphate-buffered saline(PBS) to eliminate nocodazole before release into fresh medium. Cellswere harvested every two hours and cell lysates and histones wereprepared using well-established methods (Wang, et al. (2001) Science293:853-857). The cell cycle position of the cells collected atdifferent stages was determined by propidium iodide (PI) staining. Forsimultaneous measurement of DNA content and levels of specific proteins(Mi2 and mK79), asynchronized HeLa cells were fixed by ice-cold ethanolbefore treatment with 0.25% TRITON® in PBS for 5 minutes. Subsequently,the cells were labeled with anti-Mi2 or anti-mK79 antibodies followed byfluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondaryantibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.). Thecells were labeled with PI before performing flow cytometry analysisusing stand methods.

Example 2 Results

HMTase-Mediated Methylation of H3-Lysine 79

Identification of H3-K79 as a Novel Methylation Site.

Histone methylation has emerged as an important player in regulatinggene expression and chromatin function (Jenuwein and Allis (2001)Science 293:1074-1080; Zhang and Reinberg (2001) Genes Dev.15:2343-2360; Kouzarides (2002) Curr. Opin. Genet. Dev. 12:198-209).Histone methylation occurs on arginine and lysine residues at theN-terminal tails of histones H3 and H4, and is catalyzed by two distinctfamily of proteins, the PRMT1 and the SET-domain-containing family ofproteins (Zhang and Reinberg (2001) Genes Dev. 15:2343-2360). Since thediscovery of the first histone lysine methyltransferase (Rea, et al.(2000) Nature 406:593-599), other lysine methyltransferases thatmethylate histone H3 at lysines 4, 9, 27, and 36 have been reported(Briggs, et al. (2001) Genes Dev. 15:3286-3295; Roguev, et al. (2001)EMBO J. 20:7137-7148; Wang, et al. (2001) Mol. Cell 8:1207-1217;Tachibana, et al. (2001) J. Biol. Chem. 276:25309-25317; Yang, et al.(2002) Oncogene 21:148-152; Nagy, et al. (2002) Proc. Natl. Acad. Sci.USA 99:90-94; Bryk, et al. (2002) Curr. Biol. 12:165-170; Nishioka, etal. (2002) Genes Dev. 16:479-489; Strahl, et al. (2002) Mol. Cell Biol.22:1298-1306). One common feature of these histone lysinemethytransferases is that they all contain a SET domain that is requiredfor their enzymatic activity. Thus, SET domain is believed to be asignature motif for histone lysine methyltransferase (Zhang and Reinberg(2001) Genes Dev. 15:2343-2360).

Methylation sites are known to be located at the N-terminus of histonesH3 and H4 (Jenuwein and Allis (2001) Science 293:1074-1080; Zhang andReinberg (2001) Genes Dev. 15:2343-2360). Mass spectrometry analysis hasbeen used to identify potential novel modification sites in the histoneglobular domain (Feng et al., (2002) Current Biology 12:1052-1058; thedisclosure of which is incorporated by reference herein it itsentirety). These investigations demonstrated that histone H3 from HeLacells is mono-methylated on K79 (Feng, et al., Id at FIG. 1A, panelsa-c).

Based on the well-established nucleosome structure (Luger, et al. (1997)Nature 389:251-260), K79 is located in a loop connecting the first andthe second alpha helixes (FIG. 1A). This region is exposed and isadjacent to the interface between H3/H4 tetramer and the H2A/H2B dimer.Although K79 is not directly involved in the formation of the interface,it is in a position capable of influencing the access to the interface,indicating that methylation on K79 may play an important role inregulating the access of protein factors to chromatin.

H3-K79 Methylation is Conserved from Yeast to Human.

To confirm the MS data and the conservation of K79 methylation in otherorganisms, we generated a polyclonal antibody against a dimethyl-K79 H3peptide ile-Ala-Gln-Asp-Phe-^(m)Lys-Thr-Asp-Leu-Arg-Phe (SEQ ID NO:4).To examine the specificity of the antibody, recombinant histone H3 thatwas either not methylated, or methylated on K4 or K9 by SET7 (Wang, etal. (2001) Mol. Cell 8:1207-1217) or SUV39H1 (Rea, et al. (2000) Nature406:593-599), respectively, were subjected to COOMASSIE® staining andwestern blot analysis. As a positive control, equivalent amounts of corehistones purified from HeLa cells were also analyzed. Results shown inFIG. 1B demonstrate that while the antibody recognized histone H3purified from HeLa cells, it did not recognize unmethylated, or K4-, orK9-methylated H3 (FIG. 1B, lower panel). In addition, competition withtwo peptides of identical amino acid sequences with or withoutdi-methylation on K79 demonstrate that only the methylated peptide iscapable of abolishing the reactivity of the antibody toward the HeLahistone H3. Thus, it was concluded that the antibody isH3-mK79-specific.

The fact that K79 and adjacent sequences of H3 are conserved indifferent organisms prompted us to test the possibility of H3-K79methylation occurs in other organisms. Thus, core histones isolated fromchicken, Drosophila, and budding yeast were analyzed by western blottingusing the H3-mK79-specific antibody. As negative and positive controls,recombinant H3 and HeLa core histones were included in the assay.Results shown in FIG. 1C demonstrate that H3-K79 methylation occurs inall the organisms analyzed. Thus, it was concluded that H3-K79methylation is conserved from yeast to human.

Human DOT1-Like Protein is an H3-K79-Specific Methyltransferase.

Having demonstrated that H3-K79 methylation occurs in yeast, weidentified the responsible enzyme in this organism. It was reasoned thatdeletion of the K79 HMTase should lead to loss or reduction of K79methylation that could be detected by western blot analysis using themK79-specific antibody described above. The candidate genes that wescreened included the six yeast SET domain-containing proteins (Set 1 toSet 6; a gift from Kevin Struhl and Huck Hui Ng, Harvard Medical School)as well as those that were predicted to encode SAM-bindingdomain-containing proteins (Niewmierzycka and Clarke (1999) J. Biol.Chem. 274:814-824; Dlakic (2001) Trends Biochem. Sci. 26:405-407). Thisscreen resulted in the identification of DOT1 as the HMTase responsiblefor K79 methylation in yeast (Ng, et al. (2002) Genes Dev. 16:1518-27).

Dot1 was originally identified in a genetic screen for genes whoseoverexpression disrupts telomeric silencing (Singer, et al. (1998)Genetics 150:613-632). Disruption or overexpression of Dot1 not onlyimpaired telomeric silencing, but also reduced silencing at mating typeand rDNA loci (Singer, et al. (1998) Genetics 150:613-632). In additionto participating in silencing, DOT1 also plays an important role inmeiotic checkpoint control (San-Segundo and Roeder (2000) Mol. Biol.Cell 11:3601-3615). Given that K79 methylation was conserved from yeastto human (FIG. 1C), we expected that DOT1-like (DOT1L) proteins existedin other organisms. A BLAST search revealed several putative proteinswith significant sequence homology to DOT1. Sequence alignment of theseproteins revealed several conserved blocks (FIG. 2A) that were predictedto be involved in SAM binding (Dlakic (2001) Trends Biochem. Sci.26:405-407). The BLAST search also revealed that a hypothetical humanprotein (GENBANK accession number AAC08316) encoded by a gene located on19p13.3 is likely the human DOT1L. However, this hypothetical proteinwas incomplete at both 5′ and 3′ ends. To clone the full-length cDNAencoding hDOT1L, several EST clones were obtained and sequenced. Twooverlapping EST clones (BF507396 and BF982417) contained a single openreading frame (ORF) which was predicted to encode a 1537 amino acidprotein with a calculated mass of 165 kDa (FIG. 28). The fact that thenucleotide sequence (FIG. 2C-E) around the first methionine conformed tothe Kozak initiator sequence in combination with the fact that the cDNAcontained an upstream stop codon indicated that the 1537 amino acidsencoded by the cDNA represented the full-length protein. Analysis of thehDOT1L protein sequences revealed no known functional motif other than aputative SAM-binding domain (FIG. 2B).

To determine whether hDOT1L possessed intrinsic HMTase activity, tworecombinant proteins corresponding to the N-terminal 351 and 472 aminoacids of hDOT1L, respectively, were produced in E. coli. The N-terminusof hDOT1L was selected because this region contained the putativeSAM-binding motif and was most conserved among the different DOT1Lproteins (FIG. 2A). HMTase assays revealed neither protein possessedsignificant enzymatic activity when free core histones were used as asubstrate (FIG. 3A, lanes 1-3). However, when nucleosomes were used as asubstrate, hDOT1L(1-472) exhibited significant HMTase activity whilehDOT1L(1-351) was inactive (FIG. 3A, lanes 4 and 5). To demonstrate thatthe HMTase activity depended on binding to SAM, we generated a mutatedversion of hDOT1L(1-472) [hDOT1L(1-472)m] by changing the highlyconserved Gly-Ser-Glyes-₁₆ sequence of motif I (FIG. 2A) to Arg-Cys-Arg.This mutation completely abolished the HMTase activity (FIG. 3A, comparelanes 5 and 6). Taken together, these results demonstrate that hDOT1L isa nucleosomal H3-specific HMTase and that both the SAM binding motif andthe sequences between amino acids 351 and 472 are critical for enzymaticactivity.

Having demonstrated the HMTase activity of human DOT1L in vitro, wedemonstrated its HMTase activity in vivo. Accordingly, mammalianexpression vectors encoding a FLAG®-tagged hDOT1L and a motif I mutantwere transfected into 293T cells. Core histones purified from thetransfected cells were analyzed by COOMASSIE® and western blot analysisusing antibodies specific for methylated K4, K9 or K79. Results shown inFIG. 3B demonstrate that overexpression of hDOT1L significantlyincreased H3-K79 methylation while having no effect on K4 and K9methylation (compare lanes and 2). Increased K79 methylation wasdependent on an intact motif I as transfection of a motif I mutant didnot affect K79 methylation (FIG. 3B, compare lanes 1 and 3). Thisdifferential effect was not caused by differential expression since bothconstructs expressed hDOT1L at a similar level (FIG. 3B, lower twopanels). Based on these results, we concluded that hDOT1L is anH3-K79-specific HMTase in vivo.

Methylation on K79 is Regulated During the Cell Cycle.

Total histone methylation level is regulated during the cell cycle(Borun, et al. (1972) J. Biol. Chem. 247:4288-4298). Thus, it wasdetermined whether the level of K79 methylation changed during the cellcycle. To obtain a population of synchronous cells, we arrested HeLacells at the G1/S border using a double thymidine block. After releasingthe arrested cells from the thymidine block, cells were collected everytwo hours for flow cytometry analysis as well as for the preparation ofprotein extracts and histones. Flow cytometry analysis (FIG. 4A)indicated that more than 95% of the cells progress through S phase andenter G2 synchronously. The cells were successfully arrested at the G1/Sborder before release as evidenced by the accumulation of the histonemRNA stem-loop-binding-protein (SLBP) (FIG. 4B). As has beendemonstrated (Whitfield, et al. (2000) Mol. Cell Biol. 20:4188-4198),SLBP levels stayed high throughout S phase and dropped rapidly as cellsexited S phase (FIG. 4B). The cells completed mitosis about 12 hoursafter release, as evidenced by the degradation of cyclin A when cellsentered anaphase (FIG. 4B). To determine whether the level of K79methylation changed during the cell cycle, histones isolated fromcorresponding cells were subjected to western blot analysis using themK79-specific antibody. This analysis demonstrated that the level of K79methylation decreased during S-phase and reached the lowest level in G2and increased during M-phase (FIG. 4B).

To analyze K79 methylation status in G1 phase, we arrested the cellswith thymidine, released them, and then arrested them in mitosis withnocodazole. After release from the nocodazole block, the cellsprogressed through G1 to S phase synchronously (FIG. 4C). The cellscompleted mitosis 2 hours after release from the nocodazole block asevidenced by the degradation of cyclin A (FIG. 4D). The cells thenstarted to enter S phase about 10 hours after release as evidenced bythe accumulation of SLBP (FIG. 4D). Western blot analysis of thehistones from cells collected at different time points after thenocodazole release indicated that the K79 methylation remained at a highlevel throughout the G1-phase (FIG. 4D).

To further confirm the cell cycle-dependent changes in K79 methylation,we performed bivariate analysis of DNA content and mK79 level using amethod similar to that used in analyzing cell cycle-dependent changes ofcyclins using asynchronous cells (Bonifacino, et al. (1999) CurrentProtocols in Cell Biology (John Wiley & Sons, Inc.). Book chapter 8.Unit 8.4; Darzynkiewicz, et al. (1999) Determining Cell Cycle Stage byFlow Cytometry (John Wiley & Sons, Inc.), 8.4.1-8.4.18). For eachindividual cell, DNA was labeled with propidium iodide (red), whileK79-methylated-H3 was labeled with an FITC-conjugated secondary antibody(green). The intensities of green color and red color of a particularcell reflected K79 methylation level and DNA content, respectively.Negative controls without a primary antibody or with antibody againstMi-2, a component of a nucleosome remodeling and deacetylase complex(Zhang, et al. (1998) Cell 95:279-289), resulted in a slight increase inFITC signals when cells proceeded from G1-phase to G2/M phase (FIG. 4E,left and middle panels). This result was anticipated as cell sizebecomes bigger during this transition. However, labeling of the cellswith the mK79-specific antibody resulted in a decrease in FITC-labelingwhen cells went through S-phase (FIG. 4E, right panel, compare early andlate S phases). It is unlikely that the decrease in mK79 level duringS-phase was caused by ‘dilution’ by nascent histones during chromatinassembly as a similar decrease was not observed in other methylationsites. By using two different methods, we demonstrated that the level ofK79 methylation decreased during S-phase, reached its lowest level inG2, increased during M-phase, and was maintained at a high level duringG1-phase.

Example 3 Experimental Procedures

Structure of the Catalytic Domain of Human DOT1L

Protein Methods.

To produce recombinant hDOT1L fragments containing the N-terminal 351,370, 375, 385, 416, 452, and 472 amino acids in E. coli, correspondingcDNA fragments were amplified by PCR and cloned into a pGEX-KG vector(between EcoRI-XhoI sites). The presence of correct inserts was verifiedby DNA sequencing. The plasmid expressing the first 416 residues ofhDOT1L, pGEX-KG/hDOT1L(1-416), was used for subsequent Δ(376-390) andΔ(390-407) deletions and N241D, N241A, Y312F and Y312A point mutations.The deletion and point mutations were carried out by PCR and confirmedby sequencing. GST-hDOT1L fusion proteins and mutants were firstpurified on a glutathione-SEPHAROSE® column. The GST-tag was thenremoved by thrombin digestion in the column. The eluted proteins werefurther purified on HITRAP-SP and SUPERDEXT-75 columns (AmershamPharmacia Biotech, Piscataway, N.J.).

Histone Methyltransferase Assay.

Histone methyltransferase assay was performed using established methods(Wang, et al. (2001) Science 293:853-857). Oligo-nucleosomes purifiedfrom HeLa cells or chicken blood cells were quantified and equalconcentrations were used in each assay. Briefly, recombinant hDOT1Lproteins (0.75 μM) were incubated with oligo-nucleosomes (1.5 μM) inreactions containing 20 mM Tris-HCl (pH 8.0), 4 mM EDTA, 1 mM PMSF, 0.5mM DTT, and 0.5 μL ³H-SAM (15 Ci/mM; NEN™ Life Science Products) for 1hour at 30° C. For peptide competition, 1.5 μM, 15 μM, or 150 μM ofpositively charged C-RS10 peptide(Cys-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser-Arg-Ser;SEQ ID NO:20) were added to reactions. Reactions were stopped byaddition of 7 μL of 5×SDS loading buffer and proteins were separated inan 18% SDS-PAGE. Following stain and destain of COMMASSIE® Blue, gelswere treated with Rapid Autoradiography Enhancer (NEN™ Life ScienceProducts, Boston Mass.) for 40 minutes, dried, and exposed to X-rayfilms. For quantitation, gel slices were excised and counted withscintillation counting. Two independent experiments were performed atthe same time and average ³H (CPM) values were used in the figures.

Gel Shift Assay.

Different amount of recombinant hDOT1L proteins were incubated with HeLamono-nucleosomes (1.5 μM) under the same conditions as those in thehistone methyltransferase assay, except that SAM is omitted. Followingincubation for 1 hour at 30° C., samples were resolved in a 1.8% agarosegel and visualized under UV by ethidium bromide staining.

Example 4 Results Structure of the Catalytic Domain of Human DOT1L

Disordered C-Terminal Region is Important for Enzyme Activity andNucleosome Binding.

The full-length hDOT1L consists of 1537 amino acids (Example 2) but onlythe N-terminal ˜360 amino acids share significant sequence similaritywith yeast Dot1 (FIG. 2A). We determined that amino acids 1-416 ofhDOT1L, hDOT1L(1-416), contain the active HMTase catalytic domain (FIG.5).

The crystal structure of hDOT1L(1-416) complexed with SAM has beensolved to 2.5 Å resolution using multiwavelength anomalous diffraction(MAD) of SeMet protein crystals (Min, et al. (2003) Cell 112:711-723;the disclosure of which is incorporated herein by reference). Atomiccoordinates for the hDOT1L(1-416) structure have been deposited in theRCSB database under ID code INW3). Amino acids C-terminal to αK (aminoacids 333-416) are disordered in the crystal structure. As disclosedabove, we demonstrated that certain amino acids located between residue351 and 416 were important for the enzymatic activity of hDOT1L, ashDOT1L(1-416) could methylate oligo-nucleosomes efficiently buthDot1(1-351) could not under the same conditions (FIG. 5). Since theC-terminal end of hDOT1L(1-416) was spatially distant from the SAMbinding site and because access to the active site was rather restricted(Min, et al. (2003) Cell 112:711-723), the C-terminus might be importantin substrate binding rather than having a direct involvement incatalysis. Accordingly, we tested the enzymatic activity (FIG. 6A) andnucleosome binding capability (FIG. 6B) of a series of hDOT1L variantstruncated at the disordered C-terminal region.

To identify the elements within the region between amino acids 351 and416 that were critical for the HTMase activity of hDOT1L, we added back19, 24 and 34 amino acids to the C-terminus of hDOT1L(1-351), termedhDOT1L(1-370), hDOT1L(1-375) and hDOT1L(1-385), respectively, todetermine whether the HMTase activity of hDOT1L could be restored. FIG.6A (lanes 2, 3 and 4) shows that all three constructs failed to restorethe HMTase activity to a level comparable to that of hDOT1L(1-416). Wethen fused the last 26 amino acids of hDOT1L(1-416) to the inactivehDOT1L(1-375), creating an internal deletion mutant Δ(376-390). FIG. 6A(lane 6) shows that this mutant was as active as the wild-typehDOT1L(1-416), indicating that amino acids 391-416 were crucial for theHMTase activity. This region of hDOT1L is enriched with positivelycharged residues (FIG. 6C), and these residues may be important for theHMTase activity. Removing a stretch of amino acids containing 9 of the15 positively charged residues in this region, Δ(390-407), significantlyreduced the enzymatic activity (FIG. 6A, lane 7).

We next examined mono-nucleosome binding of hDot1(1-416) and several ofits truncation variants by gel mobility shift assays (GSMA). FIG. 6Bshows that enzymatically active hDOT1L(1-416) and Δ(376-390) causedslower migration of mono-nucleosomes (lanes 2-4 and 5-7), while weaklyactive Δ(390-407) and inactive hDOT1L(1-385) showed no detectable gelshifts within the sensitivity limit of the assay. The addition of SAM inthe GSMA did not change the gel shift pattern in FIG. 68B. Thisobservation qualitatively correlated the nucleosome binding ability withthe HMTase activity of hDOT1L proteins and showed the importance of thepositively charged C-terminal region in nucleosome binding. Our modelingof hDOT1L-nucleosome interaction indicated that the C-terminal regionwas near DNA and the disordered C-terminal positively charged region maybe involved in substrate binding by interacting with the negativelycharged nucleosomal DNA. Consistent with this, we could detect strong,but apparently non-sequence specific, binding of hDOT1L(1-416) with DNAextracted from mono-nucleosomes. To further probe the nature ofhDOT1L-nucleosome interaction, we used a 21-residue highly chargedpeptide, C-RS₁₀, containing 10 tandem repeats of arginine and serine asa competitor in our nucleosome binding and HMTase assays. Adding theC-RS₁₀ peptide in GSMA prevented mono-nucleosomes from entering the gelwell. The HMTase activities of hDOT1L(1-416) and Δ(376-390), shown inlanes 1-4 and 5-8 of FIG. 6D, respectively, were effectively competedwith increasing concentrations of the peptide. These results clearlydemonstrate the functional importance of the C-terminal charged regionof hDOT1L(1-416).

Example 5 Experimental Procedures Characterization of hDOT1LLocalization

Constructs.

Full-length FLAG®-hDOT1L are described in Example 1. For FLAG®-taggedhDOT1L deletion mutants, PCR-amplified inserts were ligated to pFLAG®-beta-cDNA3 vector digested with EcoRI and XhoI. When constructingGFP-fused plasmids, all hDOT1L deletion mutants were constructed bycloning the inserts into the EcoRI and KpnI sites of pEGFP-C3 vector(CLONTECH™, Palo Alto, Calif.). For fragments larger than 20 aminoacids, PCR was used to amplify the inserts. For fragments less than 20amino acids, DNA were synthesized and annealed before ligation to thevector. For the construction of the hDOT1L-767-3×NLS construct, hDOT1cDNA encoding amino acid 1-767 was fused with three tandem repeats of anuclear localization signal (NLS) derived from SV40 T-antigen(Asp-Pro-Lys-Lys-Lys-Arg-Lys-Val; SEQ ID NO:21), and subcloned intopcDNA3b-FLAG® vector. All fragments amplified by PCR and all constructsinvolving mutagenesis were verified by sequencing.

Cell Culture, Transfection, and Immunoprecipitation.

293T or U2OS cell were maintained in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum and grown at 37° C., 5%CO₂. Cells were transfected with QIAGEN® EFFECTENE™ TransfectionReagents. Twenty-four hours after transfection, cells were harvested andwashed with ice-cold phosphate-buffered saline (PBS) before being lysedwith lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% NP-40, 50 mM NaF, 1 mMdithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 μg ofaprotinin/mL, 0.5 μg of leupeptin/mL, and 0.7 μg of pepstatin/mL). Afterincubation for 30 minutes at 4° C., the cell debris was removed bycentrifugation at 14,000 rpm for 5 minutes in an EPPENDORF® centrifuge(Brinkmann Instruments, Inc., Westbury, N.Y.). About 1 mg of proteinextracts was incubated with 20 μL of FLAG®-M2 agarose beads (Sigma, St.Louis, Mo.) at 4° C. for 4 hours. After three washes with lysis buffercontaining 300 mM salt, the immunoprecipitated proteins were analyzed bywestern blot.

In Vivo Ubiquitination Assays.

pcDNA3b-FLAG®-hDOT1L and pcDNA3b-FLAG®-Tat were transfected to 293Tcells with or without pHA-Ubiquitin (Ub) by using FUGENE™ 6 transfectionreagent (Roche Diagnostics, Indianapolis, Ind.). Twenty-four hours aftertransfection, MG132 was added at the final concentration of 25 mM, andthe cells were cultured for more hours. Cells were then lysed with lysisbuffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1% SDS, and 1 mM DTT), andboiled for 10 minutes before centrifugation at 14,000 rpm for 10minutes. The supernatant was diluted by 10-fold with NP40 lysis buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP40, 50 mM NaF, 10%glycerol, 1 mM DTT, and 1 mM PMSF), and incubated with 1.5 mg of FLAG®(M2) antibody for 4 hours at 4° C. Protein G beads were then added andincubated further for one hour. The beads were washed three times withlysis buffer containing 0.5% of NP40 before eluting the protein with SDSsample buffer for SDS-PAGE analysis.

Immunofluorescence Staining.

U2OS cells (4×10⁴) were seeded in each well of a 12-well plate one daybefore transfection. 0.3 μg DNA was used for each transfection byQIAGEN® EFFECTENE™ Transfection Reagents. Twenty-four hours aftertransfection, cells were washed twice with PBS and then fixed in 3%paraformaldehyde (in PBS) for 10 minutes at room temperature. Afterwashing once with PBS, the cells were incubated with 0.2% TRITON® X-100(in PBS) for 5 minutes at 4° C. Then the cells were blocked with 0.5%bovine serum albumin (in PBS) for 30 minutes and stained with mouseanti-FLAG® M2 antibody (IBI, Napa, Calif.; 1:1,000) or rabbit anti-HApolyclonal antibody (Abcam, Cambridge, Mass.) for 1 hour at roomtemperature. After three washes with PBS, the cells were first incubatedwith rhodamine (or FITC)-conjugated Donkey anti-mouse (or rabbit)immunoglobulin G (IgG) (Jackson ImmunoResearch Laboratories, West Grove,Pa.) diluted 1:50 with 0.5% bovine serum albumin in PBS for 30 minutesand then stained with 1 μg of 4′,6′-diamidino-2-phenylindole (DAPI)/mL(Sigma, St. Louis, Mo.) for 10 seconds. The cells were then washed threetimes with PBS, mounted with DAKO fluorescent mounting medium, andvisualized by a Zeiss immunofluorescence microscope.

Example 6 Results Characterization of hDOT1L Localization

Rapid In Vivo Degradation of hDOT1L.

The in vivo function of hDOT1L was analyzed using a rabbit polyclonalantibody generated against an N-terminal fragment (amino acids 1-351) ofhDOT1L. The affinity-purified antibody recognized 3 ng of recombinantGST-hDOT1L(1-351) (FIG. 7A, lane 6) but does not recognize any proteinbands around the expected size of about kDa either in nuclear extractsor nuclear pellet proteins derived from HeLa nuclei (FIG. 7A, lanes 4and 5). Although two specific protein bands below and above the 97 kDamarker were detected in the protein fraction derived from nuclear pellet(FIG. 7A, compare lanes 2 and 5), purification and identification ofthese proteins indicated that they were not hDOT1L (data not shown). Thefact that we could not detect endogenous hDOT1L in spite of theextensive effort, indicated that either hDOT1L existed at very lowabundance or it was subjected to rapid degradation in cells. To examinethe stability of hDOT1L, two constructs expressing an N-terminalFLAG®-tagged hDOT1L or a C-terminal HA-tagged hDOT1L were transientlyexpressed in 293T cells. The cytosol and nuclear extracts derived fromthe transfected cells were subjected to western blot analysis usingFLAG® and HA antibody, respectively. Results shown in FIG. 7B indicatethat hDOT1L was subjected to rapid degradation as numerous protein bandswere recognized by FLAG® antibody, and less than half of theFLAG®-hDOT1L existed in the full-length form although mixtures ofproteinase inhibitors were included during the preparation of theprotein extracts. Interestingly, almost no full-length FLAG®-hDOT1Lprotein was detected in the cytosol while shorter forms of FLAG®-hDOT1Lproteins detected by FLAG® antibody were comparable to that in thenuclear extracts (FIG. 7B, compare lanes 1 and 2). This indicated thathDOT1L was more rapidly degraded in the cytosol than in the nucleus.Furthermore, the HA antibody that recognized the C-terminal tag of therecombinant protein detected much fewer protein bands indicating thatthe C-terminus of the protein was extremely labile.

Subsequently, the stability of the hDOT1L and its subcellularlocalization was analyzed using hDOT1L simultaneously tagged with anN-terminal FLAG® and a C-terminal HA. After transfection, cells wereimmunostained with antibodies against FLAG® and HA, respectively (FIG.7C). Analysis of the transfected cells indicated that the great majority(>80%) of hDOT1L containing the C-terminal HA tag was localized tocytosol, while the majority (about 60%) of hDOT1L containing theN-terminal FLAG® tag was localized to the nucleus. This result isconsistent with the observation that full-length hDOT1L is mainlyobserved in nuclear fractions, while partial hDOT1L protein is enrichedin cytosol fraction. This indicates that degradation of hDOT1L mayrequire export of hDOT1L out of the nucleus.

Ubiquitination of hDOT1L.

Protein ubiquitination is the major pathway for rapid proteindegradation (Pickart (2001) Annu. Rev. Biochem 70:503-533). Toward thisend, we asked whether hDOT1L is subjected to ubiquitination. Wetransfected 293T cells with a construct encoding FLAG®-hDOT1L with orwithout co-transfection of a construct encoding HA-tagged ubiquitin.After immunoprecipitation with anti-FLAG® antibody, theimmunoprecipitates were subjected to western blot analysis using anti-HAantibodies. To ensure that FLAG®-hDOT1L was successfullyimmunoprecipitated, the immunoprecipitates were also blotted withanti-FLAG® antibody (FIG. 8A, bottom panels). Results shown in FIG. 8Aindicate that FLAG®-hDOT1L was ubiquitinated in the presence of HA-Ub(top panel, compare lanes 3 and 4). However, the presence of proteasomeinhibitor MG132 successfully stabilized ubiquitinated Tat (FIG. 8A, toppanel, compare lanes 6 and 7), but did not appear to stabilize theubiquitinated form of FLAG®-hDOT1L (FIG. 8A, compare lanes 4 and 5),indicating that hDOT1L ubiquitination was not directly linked to itsstability.

To analyze the potential function of hDOT1L ubiquitination, hDOT1Lubiquitination sites were mapped using the same approach describedabove. Different FLAG®-tagged hDOT1L deletion constructs wereco-transfected with HA-Ub and the ubiquitination of the different hDOT1Lmutants was detected by western blot analysis of HA afterimmunoprecipitation with the anti-FLAG® antibody. These analyses,summarized in FIG. 8B, allowed us to map the ubiquitination sites to aregion between amino acids 469-756 of hDOT1L.

As demonstrated below, hDOT1L(1-767) is localized to cytosol. Todetermine whether ubiquitination of hDOT1L requires cytoplasmlocalization, we artificially prevented its cytoplasmic distribution byadding three NLSs to this construct and compared its ubiquitinationlevel with a construct lacking these signals. Immunostaining oftransfected cells (data not shown) confirmed that addition of the threeNLSs efficiently prevented nuclear export of hDOT1L(1-767). However,western blot analysis of hDOT1L(1-767) with or without the three NLSsindicated that they had similar ubiquitination levels (FIG. 8C, comparelanes 5 and 6). Therefore, it was concluded that hDOT1L ubiquitinationcan occur both in the cytosol and the nucleus with similar efficiency.

Control of Cellular Localization of hDOT1L.

Given that hDOT1L is a nucleosomal histone-specific methyltransferase,we expected that hDOT1L would function in the nucleus. However, resultspresented in FIG. 7B indicated that transfected hDOT1L could localize toboth the nucleus and cytoplasm. To further verify this observation, wetransfected a plasmid encoding FLAG®-hDOT1L and visualized the proteinlocalization by immunofluorescent staining using anti-FLAG® antibody(data not shown). These studies confirmed that transiently expressedFLAG®-hDOT1L protein localized to both the nucleus and the cytoplasm.

Active nucleocytoplasmic transport plays an important role in properregulation and trafficking of nuclear proteins involved intranscription, DNA replication and chromatin remodeling (Schwoebel andMoore (2000) Essays Biochem. 36:105-113). Many of these nuclear proteinscontain both NLS and nuclear export signal (NES) sequences. In contrastto the sequences of NLS that are more conserved and could be predictedby certain software such as PredictNLS, the sequences of NES vary and noaccurate prediction is available. However, most NES sequences do sharesome common features such as a high percentage of Leucine and otherhydrophobic residues. Based on sequences of NES first identified fromHIV Rev protein and protein kinase A inhibitor (PKI), a generallyaccepted loose consensusLeu-Xaa(2,3)-[Leu-Ile-Val-Phe-Met]-Xaa(2,3)-Leu-Xaa-[Leu-Ile](SEQ IDNO:22) has been proposed. A NES database has been constructed andcontains all previously identified NESs; only 36% of the NES sequencesfit the widely accepted NES consensus (Ia Cour, et al. (2003) NucleicAcids Res. 31:393-396).

When the primary sequence of hDOT1L was examined, two potential NLSswere predicted to exist. One was located at the N-terminus and the otherat the C-terminus of hDOT1L (FIG. 9). In addition, a putativecoiled-coil domain was predicted between amino acid 516-649, whichincludes a Leucine zipper between amino acids 564-599 (FIG. 9). Thus, wemade three deletion mutant constructs of hDOT1L and examined theircellular localization by immunostaining. The N-terminal 472 amino acidsmainly exhibited nucleolar localization in U2OS cells (data not shown),which is consistent with the presence of a predicted N-terminal NLS.However, the N-terminal 767 amino acids mainly showed cytoplasmiclocalization, indicating that there was a strong NES between amino acid472 and 767, which could override the N-terminal NLS signal. The756-1537 fragment was mainly localized in the nucleus, consistent withthe presence of an NLS in this fragment.

NESs in hDOT1L.

Data presented above strongly indicated that there was at least one NESpresent between amino acids 473-767 of hDOT1L. Inspection of the aminoacid sequences in this region failed to identify sequences that matchedthe NES consensus. To identify the NES in this region, a series ofdeletion mutants of hDOT1L (469-767) constructs were fused to EGFP (FIG.10A). After transfection of these deletion mutants into U2OS cells,their cellular localization was visualized in living cells directly.Results presented in FIG. 10B indicate that there were three putativeNES sequences in this region. The first NES (NES1) resided between aminoacids and 496 having the sequencePro-Ala-Leu-Gln-Lys-Leu-Leu-Glu-Ser-Phe-Lys-Ile-Gin-Tyr-Leu (SEQ IDNO:12) (FIG. 10B, panel 12). The second NES (NES2) resided between aminoacids 600 and 635 (FIG. 10B, panel 13) and the third NES (NES3) residedbetween amino acids 636 and 650 having the sequence ofHis-Cys-Leu-Glu-Leu-Gly-Ile-Ser-Ile-Val-Glu-Leu-Glu-Lys-Ser (SEQ IDNO:13) (FIG. 10B, panel 14). Although all three NESs were able tolocalize fused EGFP to cytoplasm (FIG. 10B, panels 12-14), NES1 and NES3appeared to be much stronger than NES2. More than 95% of cellstransfected with EGFP-NES1 and EGFP-NES3 showed clear nuclear exclusion,whereas about 60% of cells transfected with EGFP-NES2 showed nuclearexclusion, with trace amounts of EGFP still remaining in nuclei. Whenthe sequences of NES1 and NES3 were compared with the NES of the humancAMP-dependent protein kinase inhibitor (PKI), several criticalhydrophobic residues were found to be conserved (FIG. 10C). While mostof the conserved amino acids were Leucine or Isoleucine, NES1 and NES3have Phenylalanine or Valine instead of Leucine at one of the conservedresidues. Previous studies indicate that C-terminal hydrophobic residuesin NES sequences are more important for nuclear export function thanN-terminal Leucines (Zhang and Xiong (2001) Science 292(5523):1910-5).To examine whether this was true for NES1, both the Phenylalanine andthe Isoleucine of NES1 were mutated to Alanine. It was found thatC-terminal hydrophobic residues were important for NES function asmutations on the two residues completely abrogated the nuclear exclusionability of NES1 (FIG. 10C). While the NES1 itself was capable oflocalizing EGFP to cytoplasm, it was important to demonstrate that NES1was functional in nuclear exclusion in the context of full-lengthhDOT1L. Thus, we examined the localization of a full-length hDOT1L NES1mutant and found that mutation of NES1 prevented cytoplasm localizationof hDOT1L (FIG. 10D) indicating that NES1 is important for hDOT1Lnuclear export.

To further characterize the nuclear export ability of the three NESsidentified, it was determined whether the NESs were involved in activenuclear export mediated by the CRM1 (chromosomal region maintenance)pathway. CRM1 was initially identified as a S. pombe protein whosemutation affects higher order chromosome structure (Adachi and Yanagida(1989) J. Cell Biol. 108:1195-1207). Subsequent studies indicate thatCRM1 is an export receptor for Leucine-rich nuclear export signals(Fornerod, et al. (1997) Cell 90:1051-1060; Ossareh-Nazari, et al.(1997) Science 278:141-144; Stade, et al. (1997) Cell 90:1041-1050).Leptomycin B (LMB), a Streptomyces metabolite, has been shown to becapable of preventing CRM1-mediated active nuclear export throughcovalent attachment to a Cysteine residue in the central region of CRM1(Kudo, et al. (1998) Exp. Cell Res. 242:540-547; Nishi, et al. (1994) J.Biol. Chem. 269:6320-6324). To investigate the effect of LMB treatmenton the nuclear export ability of the NESs, U2OS cells were transfectedwith constructs encoding each of the three NESs fused with EGFP in thepresence or absence of 20 nM of LMB. Eight hours after LMB treatment,green fluorescence was observed under a microscope in living cells.Results shown in FIG. 10E demonstrate that LMB treatment preventednuclear export by all the three NESs. These results indicate that hDOT1Lnuclear export is mediated through the CRM1 pathway.

Example 7 Experimental Procedures hDOT1L and MLL-AF10-MediatedLeukemogenesis

Constructs.

For yeast two-hybrid screening, the coding sequence of hDOT1L was clonedin-frame with the Gal4 DNA-binding domain in the pGBKT7 plasmid. For themammalian two-hybrid assay (CLONTECH™, Palo Alto, Calif.), variousregions of hDOT1L were cloned into the pM vector (CLONTECH™) to generatefusions with the Gal4 DNA-binding domain. Various regions of AF10 werecloned into the pVP16 vector (CLONTECH™) to generate fusions with theVP16 transcriptional activation domain, pG5LUC is a reporter vectorwhich contains the luciferase coding region downstream of the minimalpromoter of the adenovirus E1b gene and five GAL4 binding sites. Forretroviral vector construction, 5′ LTR region of murine stem cell virusvector (MSCVneo, CLONTECH™) were replaced by cytomegalovirus immediateearly promoter sequences (MSCN). MSCN-MLL(N) and MSCN-MLL-AF10 wereconstructed from MSCV-5′MLL and MSCV-MLL-AF10 with a FLAG®-tag upstreamof MLL gene. MSCN-hDOT1L constructs were constructed by inserting ahDOT1L cDNA (encoding amino acids 1-416 and 1-670, respectively)downstream of MLL gene. MSCB vector was constructed by the replacementof neomycin-resistant gene of MSCN with the blasticidin-resistant gene,and insertion of the full-length of hDOT1L (encoding amino acids1-1,537) downstream of a FLAG®-tag.

Yeast Two-Hybrid Screen.

To identify hDOT1L interacting proteins, yeast two-hybrid screening wasperformed with the MATCHMAKER™ Gal4 two-hybrid system 3 (CLONTECH™). ThecDNA encoding the full-length hDOT1L was fused in-frame to the GAL4DNA-binding domain in the bait vector-pGBKT7. This construct wastransformed into Saccharomyces cerevisiae host strain AH109. APre-transformed Mouse Testis MATCHMAKER™ cDNA Library (CLONTECH™) wasscreened by mating with the bait strain in accordance with themanufacturer's instructions. Approximately 4.8×10⁶ individual cloneswere screened and about 53 clones grew on the selected medium lackingHis, Ade, Trp and Leu. The clones were further selected by growth onSD/-Ade/-His/-Leu/-Trp/X-a-Gal master plates. The prey plasmids wererescued and electroporated into E. coli strain Top10. The DNA recoveredfrom the bacteria was sequenced to identify the candidate proteins thatinteract with hDOT1L.

Cell Culture, Transfections, Immunoprecipitation and Immunofluorescence.

293T and U2OS cells were maintained in DMEM supplemented with 10% fetalcalf serum and grown at 37° C. with 5% CO₂. Cell transfection andimmunoprecipitations were conducted as described in Example 5 above.

Immunofluorescence Staining.

Performed as described in Example 5 above.

Retrovirus Preparation and Transduction.

MSCN vector containing MLL-AF10 or MLL-hDOT1L were co-transfected withpGag-pol and pVSVG to human embryonic kidney cells (293T) by a standardcalcium-phosphate method. After 48 to 72 hours of transfection, thesupernatants were collected and were used for bone marrow celltransduction as follows: 4 to 12-week old C57BL/6 mice were injectedintravenously with 5-fluorouracil (150 mg/kg), and bone marrow (BM)cells were harvested from both femurs at 5 days post-injection.Retroviral supernatants were used to transduce BM cells byspinoculation. After two derail infections, infected cells were platedinto methylcellulose cultures.

Methylcellulose Colony Assays.

Retrovirally-infected BM cells (1×10⁴) were plated in 0.9%methylcellulose (Stem Cell Technologies, Vancouver, BC) supplementedwith 10 ng/mL murine IL-3, IL-6, GM-CSF, and 50 ng/mL SCF in thepresence of 1 mg/mL G418 (GIBCO, Grand Island, N.Y.). After 7 days ofculture, the number of colonies was counted and 1×10⁴ cells of thesingle-cell suspensions prepared from G418-resistant colonies werereplated into methylcellulose supplemented with the same growth factorswithout G418. Further plating was repeated every 10 days. For theexperiment in FIG. 15, 1×10⁴ cells were plated with 5 μg/mL ofBlasticidin.

RT-PCR Analysis of Hox Genes.

Total RNA was isolated from primary bone marrow cells orMLL-AF10/MLL-hDOT1L-transduced cells using RNEASY® (QIAGEN®). Onemicrogram of total RNA was treated with RNase-free DNase I, and appliedfor reverse transcription using IMPROM-II™ (PROMEGA®, Madison, Wis.)according to manufacturer's protocol. The resulting cDNA was used astemplate for PCR amplification of Hox genes using PLATINUM® TaqPolymerase (INVITROGEN™, Carlsbad, Calif.). Primers sequences weredesigned based on the known hox gene sequences.

Mouse Transplantation and Histological Analysis.

Six-week-old non-obese/severe combined immunodeficiency (NOD/SCID) micewere injected intravenously through ophthalmic vein with 10⁶MLL-hDOT1L(1-670)- or MLL-AF10-transduced BM cells derived from the3^(rd) round of methylcellulose colonies. For tumor analysis, tumortissues were fixed in 4% paraformaldehyde. After being paraffinembedding and sectioning, slides were stained with hematoxylin and eosinusing standard techniques.

Example 8 Results hDOT1L and MLL-AF10-Mediated Leukemogenesis

AF10 Interacts with hDOT1L.

Having cloned and characterized the structure, H3-K79-specificmethyltransferase activity, and subcellular localization of hDOT1L, wesearched for functional partners by yeast two-hybrid screening. Thisscreen resulted in repeated isolation of cDNAs encoding the C-terminalhalf of AF10 (FIG. 11A), a frequent fusion partner of MLL and CALM inleukemia (Chaplin, et al. (1995) Blood 86:2073-2076; Dreyling, et al.(1996) Proc. Natl. Acad. Sci. USA 93:4804-4809). To confirm theinteraction, we co-expressed AF10 and hDOT1L, respectively tagged withFLAG® and HA. Following immunoprecipitation with anti-FLAG® antibody andwestern blot analysis with anti-HA antibody, we demonstrated that thetwo proteins can be co-immunoprecipitated (FIG. 11B, compare lanes 3 and6). In addition, the interaction was also confirmed by mammaliantwo-hybrid assays (FIG. 12 and FIG. 13). Therefore, AF10 associates withhDOT1L in vivo.

The OM-LZ Region of AF10 is Necessary and Sufficient for the AF10-hDOT1LInteraction.

AF10 was initially discovered by virtue of its involvement int(10;11)(p12;q23) chromosomal translocations found in acute myeloidleukemia patients (Chaplin, et al. (1995) Blood 86:2073-2076). Thisgenetic rearrangement results in fusion of the C-terminal half of AF10to the N-terminal third of MLL. AF10 contains several motifs common totranscription factors, and the minimal portion of AF10 fused to MLLcontains a leucine zipper (LZ) motif that is highly conserved betweenhomologs of AF10 from C. elegans to human (FIG. 12A). To characterizethe nature of the AF10-hDOT1L interaction, we mapped the region(s) ofAF10 involved in the interaction. Mammalian two-hybrid assays wereperformed in 293T cells. AF10 and hDOT1L, fused to the VP16 activationdomain and the Gal4-DBD, respectively, were co-expressed in the presenceof a luciferase reporter containing five Gal4 binding sites. Resultsshown in FIG. 12B demonstrate that a region of about 80 amino acids(amino acids 719-800) is sufficient to mediate the interaction.Interestingly, this same region has been recently demonstrated to berequired for leukemic transformation of the MLL-AF10 fusion protein(DiMartino, et al. (2002) Blood 99:3780-3785). To evaluate whether this80-amino acid region was necessary for the interaction, we created anAF10 construct where this region was deleted. FLAG®-tagged AF10 with orwithout this region was co-expressed with hDOT1L-HA and their ability tointeract with each other was analyzed by co-immunoprecipitation.Although both constructs were expressed to a similar level (FIG. 12C,left panel), deletion of this 80 amino acid region abrogated the abilityof AF10 to interact with hDOT1L (FIG. 12C, right panel, compare lanes 6and 9). Therefore, we concluded that the 80-amino acid region wasnecessary for the AF10-hDOT1L interaction.

In addition to a leucine zipper, this 80-amino acid region also includesan octapeptide motif (OM), (Glu-Gin-Leu-Leu-Glu-Arg-Gin-Trp; SEQ IDNO:19), which is highly conserved among AF10 homologs (FIG. 12A).Previous studies indicate that fusion of OM or LZ alone to MLL is notsufficient for leukemic transformation. However, fusion of MLL to OM+LZis sufficient for leukemic transformation (DiMartino, et al. (2002)Blood 99:3780-3785). To evaluate the relative contribution of OM and LZto hDOT1L interaction, we created constructs harboring mutations in thetwo regions individually or in combination and analyzed their effect onthe AF10-hDOT1L interaction using the mammalian two-hybrid assaydescribed herein. Results shown in FIG. 12D indicate that while mutationin each of the two regions greatly reduced their interaction, mutationin both regions simultaneously completely destroyed the interaction.Thus, it was concluded that both OM and LZ contributed to thehDOT1L-AF10 interaction. Collectively, these data indicate that theOM-LZ region required for leukemic transformation is both necessary andsufficient for hDOT1L-AF10 interaction.

A Leucine-Rich Region Mediates the AF10-hDOT1L Interaction.

Having defined the region in AF10 required for the AF10-hDOT1Linteraction, we mapped the region(s) in hDOT1L involved in theinteraction. Constructs encoding different regions of the hDOT1L weregenerated and co-expressed with AF10 in 293T cells. The ability of thesehDOT1L deletion mutant proteins to interact with AF10 was analyzed byco-immunoprecipitation. Results shown in FIG. 13A indicate that a regionof 294 amino acids (amino acids 473-766) to be important for theinteraction (compare lanes 8 and 10).

To confirm these results and narrow down the interaction region, themammalian two-hybrid assay described above 4 was used. Specifically, aplasmid encoding the VP16 activation domain fused to the OM-LZ region ofAF10 was co-expressed with various constructs encoding Gal4DBD fused todifferent portions of hDOT1L(472-767). Interaction was measured byactivation of a luciferase reporter containing five Gal4 binding sites.Comparison of the activation ability of the first five constructsallowed us to map the interaction domain to a region of 130 amino acids(amino acids 500-630) predicted to form a coiled-coil domain withpotential to be a leucine zipper (FIG. 13B, 1-5). Further deletion andmutational studies identified two leucine residues (L532 and L592)critical to the interaction (FIG. 13B, 6-9). The importance of these twoleucine residues in mediating the hDOT1L and AF10 interaction wasverified in the context of partial and full-length hDOT1L (FIG. 13B,10-13). Accordingly, it was concluded that a leucine rich region betweenamino acids 500-630 of hDOT1L is involved in hDOT1L and AF10 interactionand at least two of the leucine residues (L532 and L592) are criticalfor the interaction.

Immortalization of Primary Murine Myeloid Progenitor Cells byMLL-hDOT1L.

The fact that the OM-LZ domain of AF10 is both necessary and sufficientin mediating AF10 and hDOT1L interaction in combination with the factthat the same region is required for leukemic transformation (DiMartino,et al. (2002) Blood 99:3780-3785) raised the possibility thatMLL-AF10-mediated leukemic transformation may involve recruitment ofhDOT1L and its associated HMTase activity to affect expression of MLLtarget genes. Consistent with this, immunofluorescence staining revealedco-localization of transfected hDOT1L and MLL-AF10 as large nuclear foci(data not shown). If the fused AF10 in MLL-AF10 only serves to recruithDOT1L, leukemic transformation may be achieved by direct fusion ofhDOT1L to MLL. To test this, a methylcellulose serial replating assay,outlined in FIG. 14A, was used to assess immortalization of murinemyeloid progenitor cells by MLL-hDOT1L. A modified murine stem cellvirus (MSCV)-derived retroviral vector, designated as MSCN, was used totransduce freshly harvested bone marrow cells from mice pretreated with5-flurouracil. MSCN constructs encoding MLL sequences 5′ of thetranslocation breakpoint with AF10 (MLL-N), MLL-AF10 that mimic the mostfrequent fusion involving the two proteins, MLL fused to various lengthof hDOT1L, and hDOT1L were made (FIG. 14B). To facilitate expressionanalysis of these constructs, an N-terminal FLAG®-tag was added to eachof the constructs. Due to the size limitation for efficient packaging ofretroviral vectors, MLL-hDOT1L fusion constructs encoding more than thefirst 670 amino acids of hDOT1L had dramatically decreased retroviraltransduction efficiency (data not shown), which prevented us fromanalyzing MLL-hDOT1L that contained the full-length hDOT1L using thisassay. Nevertheless, retroviruses with similar titer as that of MLL-AF10were successfully generated with MLL-hDOT1L(1-416) and MLL-hDOT1L(1-670)(data not shown). As disclosed herein, hDOT1L(1-416) has robust H3-K79methyltransferase activity in vitro. Importantly, transient transfectionof MLL-hDOT1L(1-416) and MLL-hDOT1L(1-670) into 293T cells resulted inincreased H3-K79 methylation in vivo (data not shown) indicating thatboth fusion proteins were enzymatically active. Similar to thefull-length hDOT1L mutant, the two parallel constructs that harbor“GSG163-165RCR” mutations were enzymatically inactive in the same assay(data not shown). Western blot analysis of extracts fromtransiently-transfected retroviral packaging cells confirmed that MLL-N,MLL-AF10, MLL-hDOT1L(1-416), MLL-hDOOT1L(1-670), and hDOT1L constructswere efficiently expressed (data not shown). Plating of cells transducedwith the various MSCN constructs under selective conditions showed avariable number (50-100) of colonies that were consistent with theirrespective virus titer. However, significant differences were observedin a second round of plating of 10⁴ cells pooled from colonies harvestedfrom the first round of cultures. Compared to the first round, vectoralone, MLL-N, hDOT1L, and the two MLL-hDOT1L mutants transduced culturesproduced a decreased number of secondary colonies. In contrast,MLL-hDOT1L(1-416), MLL-hDOT1L(1-670) and MLL-AF10 transduced cells gaverise to hundreds of colonies, an amount significantly higher than thatfrom the first round of plating. When a third round of plating wasassayed, only MLL-hDOT1L(1-670) and MLL-AF10 transduced cells gave riseto an increased number of colonies compared with that from the secondround of plating. It was therefore concluded that similar to MLL-AF10,MLL-hDOT1L(1-670) has leukemic transformation capability. This abilitydepends on both the hDOT1L H3-K79 methyltransferase activity and theregion between amino acid 417-670.

HMTase Activity of hDOT1L is Required to Maintain the Growth Capacity ofMLL-AF10-Transformed Cells.

To further demonstrate that the enzymatic activity of hDOT1L is criticalin MLL-AF10-mediated leukemogenesis, we examined the effect of thewild-type hDOT1L and the HMTase-defective mutant hDOT1L on thesustaining growth capability of MLL-AF10-transduced bone marrow cells.Nucleic acid sequences encoding full-length wild-type andHMTase-defective mutant hDOT1L proteins were cloned into the retroviralvector pMSCB which expresses the Blasticidin resistant gene. Virusesgenerated using this vector were used to infect MLL-AF10-transduced bonemarrow cells derived from the third round of plating (FIG. 15A). Cellsexpressing hDOT1L were selected in the presence of Blasticidin. After 10days of methylcellulose culture, we observed a dramatic difference inthe colony formation assay (FIG. 15B). While transduction of wild-typehDOT1L into MLL-AF10 expressing-cells still supported colony formation,transduction of HMTase-defective hDOT1L into MLL-AF10 expressing-cellscompletely eliminated the ability of MLL-AF10 to support colonyformation. These results support the finding that MLL-AF10's ability tosustain the growth of transformed bone marrow cells is dependent uponthe HMTase activity of hDOT1L. It also indicates that theHMTase-defective hDOT1L protein can function as a dominant-negativefactor in maintaining the growth capacity of MLL-AF10 transformed cells.

Immortalization of Murine Myeloid Progenitors by MLL-hDOT1L.

While MLL-hDOT1L(1-670) is capable of transforming bone marrow cells,the number of colonies formed is different from that of MLL-AF10 (FIG.14C). In addition, the morphology of the colonies formed also appearedto be different (FIG. 16A). Similar to a previous report (DiMartino, etal. (2002) Blood 99:3780-3785), colonies arising fromMLL-AF10-transduced bone marrow cells exhibited round, compactmorphology (FIG. 16A). In contrast, MLL-hDOT1L(1-670)-transformed bonemarrow cells form colonies with two types of morphologies that weredifferent from that of MLL-AF10 transformed cells (FIG. 16A).Interestingly, the cells of type II colonies fromMLL-DOT1(1-670)-transformed cells have a significant growth advantagerelative to that of type I colonies in the third round of plating (FIG.16B). To determine the cell lineage of these colonies, immunophenotypingof these cells in early liquid cultures was performed. Consistent withmyeloid cell lineage, many cells derived from MLL-AF10-transformedcolonies or the type I MLL-hDOT1L-transformed colonies expressed theearly myeloid markers Mac-1 and c-Kit (FIG. 16C). In contrast, themajority of cells from the type II colonies of MLL-hDOT1L-transformedcells were negative for Mac-1 or c-Kit, indicating they were most likelynot of myeloid lineage (FIG. 16C). These results indicated that althoughMLL-hDOT1L(1-670) was capable of immortalizing bone marrow cellsarrested at a relatively early stage of myeloid differentiation, itcould not completely phenocopy MLL-AF10. Whether the difference wascaused by the use of partial, instead of the full-length, hDOT1L in theMLL fusion and colony assay remains to be determined.

Up-Regulation of a Specific Subset of Hox Genes in MLL-hDOT1L-MediatedMyeloid Transformation.

Similar to its Drosophila homologue Trx, mouse MLL plays an importantrole in maintaining the Hox gene expression pattern during embryogenesis(Yu, et al. (1995) Nature 378:505-508). In addition to participating inembryogenesis, Hox gene expression also plays important roles in normalhematopoiesis and leukemogenesis processes (Buske and Humphries (2000)Int. J. Hematol. 71:301-308; Sauvageau, et al. (1994) Proc. Natl. Acad.Sci. USA 91:12223-12227). To understand how hDOT1L and its associatedHMTase activity may contribute to leukemogenesis, we determined theexpression profile of Hox genes in murine primary bone marrow cellsbefore and after immortalization by MLL-AF10 or MLL-hDOT1L(1-670). Sincethese cell lines represent the earliest stages of myeloidleukemogenesis, they likely have sustained very few, if any, secondarymutations. Given that MLL-hDOT1L-transformed bone marrow cells give riseto two types of morphologically different colonies, we analyzed theirHox gene expression patterns separately. Accordingly, RNAs were isolatedfrom primary, MLL-AF10- or MLL-hDOT1L-transformed bone marrow cells.Expression of individual Hox genes in these cells was analyzed byreverse transcriptase PCR (RT-PCR). GAPDH was used as a control forequal RNA input for the different samples. Similar to a previous report(Ayton and Cleary (2003) Genes Dev. 17:2298-2307), cells immortalized byMLL-AF10 have the most numbers of genes in the HoxA complex up-regulatedrelative to that in primary bone marrow cells (FIG. 17A). Consistentwith the colony assay result demonstrating that the colonies generatedfrom MLL-hDOT1L(1-670)-transduced cells were different from thosegenerated from MLL-AF10 transduced cells, cells derived from both typesof MLL-hDOT1L(1-670)-transduced colonies had fewer numbers of Hox genesup-regulated relative to the MLL-AF10-transformed cells. Indeed, acomplete analysis of all the 39 Hox genes identified HoxA9 as the onlygene that was up-regulated in MLL-AF10 and both types ofMLL-hDOT1L(1-670)-transformed cells (FIG. 17B). These data indicate thatHoxA9 is likely to be a critical gene in leukemogenesis-mediated by MLLfusion proteins.

MLL-hDOT1L-Transduced Cells Induce Tumors in Mice.

The fact that MLL-hDOT1L can transform bone marrow cells in the colonyassay indicates that hDOT1L may be an oncogene. To evaluate thetumorgenic potential of MLL-hDOT1L, cells derived from third round ofplating were transplanted into SCID mice. Of the four mice transplantedwith MLL-hDOT1L-transformed cells, two developed terminal tumor withinfour weeks of transplantation, the other two mice still appeared healthyat five weeks after transplantation (data not shown). The control miceand the mice transplanted with MLL-AF10-transformed cells still appearedhealthy at four weeks of transplantation, consistent with previousreports that SCID mice transplanted with MLL-AF10-transformed bonemarrow cells have at least eight weeks of latency. Histological analysisof the tumor derived from transplant of MLL-hDOT1L-transformed bonemarrow cells revealed the tumor as undifferentiated tumor (data notshown).

hDOT1L Transports AF10 to the Cytosol.

Results provided herein indicate that hDOT1L participates inMLL-AF10-mediated leukemogenesis. To understand the functionalconsequence of the AF10 and hDOT1L interaction in a non-leukemicsituation, we examined the effect of their association on their cellularlocalization in view of the fact that the three NESs of hDOT1L overlapwith the region involved in the AF10 interaction. AF10 and hDOT1L,tagged with green fluorescent protein (GFP) and FLAG®, respectively,were transfected into U2OS cells and their cellular localization wasviewed by microscopy. Immunostaining with anti-FLAG® antibody revealedthat hDOT1L localized to both the nucleus and the cytoplasm whileGFP-AF10 localized to the nucleus exclusively when the two proteins wereexpressed individually (data not shown). Unexpectedly, whenco-expressed, these proteins not only co-localized, but were alsoexcluded from nucleus (data not shown), indicating that one of theconsequences of AF10 and hDOT1L association is export of both proteinsto cytoplasm. Given that the function of hDOT1L is to methylatenucleosomal histone H3, association with AF10 and subsequent transportto cytosol likely impedes the ability of hDOT1L to methylate nucleosomalH3. In addition, the nuclear function of AF10 is most likely affected byits export from nucleus. It is possible that the association of AF10with hDOT1L may expose the NLS of hDOT1L and facilitate its nuclearexport.

These results, in combination with the finding that MLL-AF10 can directthe nuclear localization of hDOT1L allowed us to propose a function forhDOT1L in normal and leukemia cells. Not to be bound by theory, AF10 ispossibly exported from nucleus when it associates with hDOT1L undernormal conditions (FIG. 18A). However, in leukemia cells, where theC-terminal part of AF10 is fused to MLL, although the MLL-AF10 fusionprotein is still capable of interacting with hDOT1L through the OM-LZpresent in the C-terminal half of AF10, the MLL-AF10/hDOT1L complexcannot export from the nucleus either because the N-terminal of AF10 ismissing or because the nuclear localization signal in MLL keeps theprotein complex from exporting (FIG. 18B). Regardless of the underlyingmechanism, one significant epigenetic change in leukemia cells relativeto that of normal cells is predictable. It has been demonstrated thatMLL is an H3-K4 methyltransferase (Milne, et al. (2002) Mol. Cell10:1107-1117; Nakamura, et al. (2002) Mol. Cell 10:1119-1128). Thepromoter of MLL target genes, for example HoxA9, are likely to beenriched for H3-K4 methylation in normal cells. However, in leukemiacells, as a consequence of MLL and AF10 fusion, the promoter of MLLtarget gene will not be enriched for H3-K4 methylation due to the lossof the MLL C-terminal SET domain. Instead, the MLL-AF10 fusion willbring in the hDOT1L resulting in H3-K79 methylation of the MLL targetgene promoter (FIG. 18A and FIG. 18B).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

The invention claimed is:
 1. A method of detecting the level ofmethylated histone H3 lysine 79 (H3-K79) associated with the HoxA9 genein a subject, comprising: obtaining a biological sample comprisingnucleosomes and the HoxA9 gene from a subject; adding to the biologicalsample an antibody that binds to a methylated histone H3 lysine 79(H3-K79); and detecting the level of H3-K79 methylation associated withthe HoxA9 gene; wherein the biological sample is a B cell sample or bonemarrow sample.
 2. The method of claim 1, wherein the biological sampleis a nucleosome preparation.
 3. The method of claim 1, wherein themethod further comprises detecting the level of histone H3 lysine 4methylation associated with the HoxA9 gene.
 4. The method of claim 1,wherein the biological sample is a B cell sample.
 5. The method of claim1, wherein the biological sample is a bone marrow sample.